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Archive for the 'Audits and Billing Issues' Category
Nitin Chhoda, A Licensed Physical Therapist Shares the Most Common Medical Billing Errors to Physical Therapy Practices
May 12, 2013 Joe Rugg Leave a comment
More and more healthcare providers have chosen to outsource their medical billing process so that they do not have to worry about committing unnecessary billing and coding errors. Licensed physical therapist, Nitin Chhoda, recently released the most common medical billing errors to practitioners in order to minimize denials on their future medical billing process.
See on www.prweb.com
Categories: Audits and Billing Issues
Recent Criminal and Civil Enforcement Actions
February 15, 2013 Joe Rugg Leave a comment
From Akerman's Health Law Rx Blog:
POSTED BY JOSEPH W. N. RUGG ON FEBRUARY 15, 2013
In 2009, the Health Care Fraud Prevention and Enforcement Action Team ("HEAT") initiative was formed as a joint effort between the Departments of Justice and Health and Human Services, and combating health care fraud continues to be a major focus of federal and state authorities. The Office of Inspector General ("OIG") publishes a list of the criminal and civil enforcement actions and the punishment that has been meted out to those convicted of some sort of healthcare fraud. Here are three examples from February listings where jail time or substantial fines or both have been imposed.
On February 1, a 50-year-old pharmacist who owned and operated 26 pharmacies in the Detroit area was sentenced to 17 years in prison. The evidence presented at trial showed that during 2006 through 2011 the pharmacies had billed Medicare and Medicaid more than $57 million, at least 25% of which billings were fraudulent as being either medically unnecessary or never actually dispensed. Similar fraudulent activities occurred with respect to commercial insurance. The pharmacist's business model was to pay kickbacks to physicians in exchange for writing prescriptions for expensive medications or for controlled substances without regard to medical necessity. In addition to the jail time, the defendant was required to repay Medicare and Medicaid $17.3 million and commercial insurers $1.5 million.
On February 4, two patient recruiters for a Miami home healthcare company pleaded guilty for their participation in a $20 million home health Medicare fraud scheme. The recruiters admitted that during 2007 through 2009, they recruited patients for which the home health agency could bill Medicare, and the home health agency's owners paid them kickbacks and bribes. Medicare was billed for home health care and therapy services that were either medically unnecessary or not provided. The recruiters face jail time and fines, and each of the two home health agency's owners have already been sentenced to over 6 years in jail.
On February 7, St. Joseph's Medical Center, a hospital located in Towson, MD, agreed to pay $4.9 million in connection with its submission of false claims to Medicare, Medicaid, and other federal healthcare programs. The hospital stated that during 2007 through 2009 it admitted patients for short stays – typically one or two days – that were not warranted by the patient's medical condition but which would result in larger insurance payments than was appropriate.
If you are aware of potentially fraudulent or even inadvertent noncompliant activities in your healthcare business, you need to seek assistance to fix those problems before they become the object of an investigation. Because former employees and business partners of healthcare providers are often the ones reporting the fraudulent activities to the authorities, problems cannot be ignored.
Since the the above blog was posted on the Akerman Health Law Rx Blog, the following was posted by the OIG:
Cardiologist admits taking cash kickbacks for patient referrals — According to a February 14, 2013 press release from the Office of the U.S. Attorney, District of New Jersey:
Shashi Agarwal, 60, of Edison, N.J., who has his own cardiology practice in East Orange, N.J., pleaded guilty before U.S. District Judge Claire C. Cecchi in Newark federal court to an Information charging him with one count of soliciting and receiving more than $100,000 in cash kickbacks in violation of the federal health care anti-kickback statute.
Agarwal is the 10th person to plead guilty in the government's investigation into the scheme to pay cash to health care providers who referred patients to Orange Community MRI, LLC (Orange MRI) in Orange N.J., for diagnostic testing.
Categories: Audits and Billing Issues, Florida, Fraud and Abuse, Healthcare Business, Hospitals, Insurance
Akerman's Health Law Rx Blog
February 4, 2013 Joe Rugg Leave a comment
I am pleased to announce my firm's new health law blog, Health Law Rx Blog.
Akerman's Health Law Rx Blog provides timely updates on the latest health law issues, keeping the firm's clients, friends, and readers up to date on pertinent legal developments. Akerman attorneys regularly update the blog with changes in the law and other relevant news. As this is meant to be an interactive site, your comments and contributions are appreciated. I am one of the contributors, so I hope you will visit the blog often and participate in any discussions that interest you. I plan to shadow post articles from the blog that I think you will find interesting.
Content on Akerman's Health Law Rx Blog is intended to inform you about legal developments, including recent decisions of various courts and administrative bodies. It should not be construed as legal advice or a legal opinion, and you should not act upon the information without seeking the advice of legal counsel.
With more than 550 lawyers and government affairs professionals and a network of 19 offices, Akerman is ranked among the top 100 law firms in the U.S. by The National Law Journal NLJ 250 (2012). The firm's Healthcare Practice Group includes over twenty attorneys and professionals representing health systems, physicians, health insurers, and other clients in all aspects of healthcare law across Florida and throughout the United States.
Categories: Akerman, Audits and Billing Issues, CMS Updates, Dentists/Orthodontists, Electronic Health Records, Florida, Fraud and Abuse, Health Law Reform -- General, Healthcare Business, Hospitals, Improving Healthcare, Insurance, Legislation, Medicaid, Medicare, Physician Practices, Regulation
USDOJ – NJ: Former director of diagnostic testing center admits bribing doctors in cash-for-patients scheme
October 20, 2012 Joe Rugg Leave a comment
In the following post, note that the dollars involved are relatively modest:
The former executive director of Orange Community MRI LLC, today admitted paying bribes to doctors since April 2008 and agreed to forfeit $89,000 in proceeds from the crime, U.S. Attorney Paul J. Fishman announced.Chirag Patel, 37, of Warren, N.J., pleaded guilty to an Information charging him with one count of soliciting and receiving illegal cash kickbacks for patient referrals in violation of the federal health care anti-kickback statute.
According to documents filed in this case and statements made in court:
On Dec. 8, 2011, Patel was arrested and charged with offering and paying cash kickbacks to a New Jersey health care practitioner in exchange for referrals to Orange Community MRI. On Dec. 13, 2011, 13 New Jersey doctors and one nurse practitioner were arrested and charged in separate Complaints with accepting similar cash kickback payments from Orange MRI. Each of the defendants was recorded taking envelopes of cash in exchange for patient referrals.
Patel is the ninth person charged in the December 2011 takedown to plead guilty. Patel is the second member of Orange Community MRI to plead guilty; Ashokkumar Babaria, the former owner and medical director of Orange Community MRI, pleaded guilty on Sept. 27, 2012.
As part of his plea agreement, Patel agreed to forfeit $89,180 that constitutes criminal proceeds of the crime. As part of his guilty plea, Ashokkumar Babaria agreed to forfeit his revenues traceable to corrupt referrals, which the government has estimated could reach as much as $2 million. The seven health care providers that referred patients to Orange MRI who have plead guilty thus far have collectively agreed to forfeit over $150,000 in illegal kickbacks from Orange MRI.
See on www.justice.gov
For an aggregation of other articles on Hot Topics in Healthcare Law, go to my magazine on Scoop.it – Hot Topics in Healthcare Law and Regulation and my newspaper on Paper.li – Hot Topics in Healthcare Law.
For an aggregation of other articles on improving healthcare, go to my internet magazine Scoop.it! Changing Health for the Better.
Categories: Audits and Billing Issues, Fraud and Abuse, Medicare, Physician Practices, Regulation, Risk Managment
USDOJ: Medicare Fraud Strike Force Charges 91 Individuals for Approximately $430 Million in False Billing
October 5, 2012 Joe Rugg Leave a comment
Medicare Fraud Strike Force operations in seven cities have led to charges against 91 individuals – including doctors, nurses and other licensed medical professionals – for their alleged participation in Medicare fraud schemes involving approximately $429.2 million in false billing, Attorney General Eric Holder and Health and Human Services (HHS) Secretary Kathleen Sebelius announced today.
Attorney General Holder and Secretary Sebelius were joined in the announcement of the nationwide takedown by Assistant Attorney General Lanny A. Breuer of the Justice Department's Criminal Division, FBI Associate Deputy Director Kevin Perkins, Inspector General Daniel R. Levinson of the HHS Office of Inspector General (HHS-OIG) and Dr. Peter Budetti, Deputy Administrator for Program Integrity of the Centers for Medicare and Medicaid Services (CMS).
"Today's enforcement actions reveal an alarming and unacceptable trend of individuals attempting to exploit federal health care programs to steal billions in taxpayer dollars for personal gain," said Attorney General Holder. "Such activities not only siphon precious taxpayer resources, drive up health care costs, and jeopardize the strength of the Medicare program – they also disproportionately victimize the most vulnerable members of society, including elderly, disabled and impoverished Americans."
"Today's arrests put criminals on notice that we are cracking down hard on people who want to steal from Medicare," said HHS Secretary Sebelius. "The health care law gives us new tools to better fight fraud and make Medicare stronger. In addition to the arrests made today, HHS used new authority from the health care law to stop future payments to many of the health care providers suspected of fraud, saving Medicare resources and taxpayer dollars from being lost to fraud in the first place."
Categories: Audits and Billing Issues, Florida, Fraud and Abuse, Medicare, Physician Practices
Manoj Jain: Doctors need to eliminate waste from healthcare — Memphis Commercial Appeal
September 23, 2012 Joe Rugg Leave a comment
Dr. Manoj Jain is an infectious disease physician and also writes for The Washington Post. His articles can be seen on MJainMD.com.
Thirty percent of health care spending — amounting to $750 billion a year — is wasted, according to a recent report by the Institute of Medicine.
I know. As a doctor, I am party to this waste, and I think doctors can play a major role in recovering it.
In a private conversation, a cardiologist tells me about his partners — "loose guns" he calls them. "At the hint of chest pain they will do a cardiac cath and this makes everyone happy," he says. The patient feels good that something was done, the doctor gains certainty of his presumptive diagnosis and the hospital makes money. While it may seem like a win-win-win, in fact, we all lose as the health care expenditure tops $2 trillion, siphoning funds from education, housing and business innovation.
The IOM report notes that unnecessary services are responsible for nearly a third, or $210 billion, of wasted expenditure.
I, too, order excessive services like CT and MRI scans, without regard to cost. Often these services are in the gray zone of medicine where it is unclear if some procedures are really necessary.
When we doctors talk about waste, we often beat around the bush. We know the system is full of waste, but when confronted we blame the patients or malpractice attorneys.
There is another less-talked-about reason for unnecessary services. One person's waste is another person's income. Another cardiac catherization, another back surgery means more income for doctors, hospitals and the health care system and its archaic administrative services.
To reduce waste, doctors need to become integral partners in the cost-cutting process.
See on www.commercialappeal.com
Categories: Audits and Billing Issues, Fraud and Abuse, Healthcare Business, Improving Healthcare, Insurance
Louisiana Couple sentenced in Medicare fraud case
A Plaquemine couple is headed to federal prison after admitting that they used their medical equipment company to defraud Medicare of slightly more than $1 million.
The couple admitted they billed Medicare for equipment that was either medically unnecessary or never delivered to Medicare beneficiaries. In some cases, Medicare was billed for expensive equipment for which inferior products were substituted.
Between January 2006 and March 2008, according to their charge, the Stewarts and "others known and unknown to the United States attorney" falsely billed Medicare for $1.9 million in power wheelchairs, orthotics and other durable medical equipment.
See on www.fox8live.com
Categories: Audits and Billing Issues, Fraud and Abuse, Medicaid
Medical Holocaust Blogpost: Florida, HCA, Rick Scott and Medicare Fraud
Some strong opinions and YouTube videos from the Medical Holocaust Blogpost:
"Rick Scott is just one of many corporate gangsters operating in the lucrative and largely unregulated medical industry. Now street level thugs in Florida have taken a lesson form guys like Rick Scott and are bilking Medicare for billions. Rick Scott wins the gold medal when it comes to bilking Medicare and private insurance out of billions and his gold medal performance has inspired other criminals of a slightly different pedigree to follow in his footsteps."
See on medicalholocaust.blogspot.fr
Categories: Audits and Billing Issues, Florida, Fraud and Abuse, Hospitals
A Harsh Reminder of How Active the OIG, DOJ, and State AGs are in Health Fraud Prevention
A harsh reminder to healthcare providers that the OIG, DOJ, and state AGs are actively prosecuting healthcare fraud.
See on oig.hhs.gov
Categories: Audits and Billing Issues, Fraud and Abuse, Medicaid, Medicare
USDOJ: Hospital Chain HCA Inc. Pays $16.5 Million to Settle False Claims Act Allegations Regarding Chattanooga, Tenn., Hospital
HCA Inc., one of the nation's largest for-profit hospital chains, has agreed to pay the United States and the state of Tennessee $16.5 million to settle claims that it violated the False Claims Act and the Stark Statute, the Department of Justice announced today.
As alleged in the settlement agreement, during 2007, HCA, through its subsidiaries Parkridge Medical Center, located in Chattanooga, Tenn., and HCA Physician Services (HCAPS), headquartered in Nashville, Tenn., entered into a series of financial transactions with a physician group, Diagnostic Associates of Chattanooga, through which it provided financial benefits intended to induce the physician members of Diagnostic to refer patients to HCA facilities. These financial transactions included rental payments for office space leased from Diagnostic at a rate well in excess of fair market value in order to assist Diagnostic members to meet their mortgage obligations and a release of Diagnostic members from a separate lease obligation.
The Stark Statute restricts financial relationships that hospitals may enter into with physicians who potentially may refer patients to them. Federal law prohibits the payment of medical claims that result from such prohibited relationships.
"The Department of Justice continues to pursue cases involving improper financial relationships between health care providers and their referral sources, because such relationships can corrupt a physician's judgment about the patient's true healthcare needs," said Stuart F. Delery, the Acting Assistant Attorney General for the Department of Justice's Civil Division.
"Physicians should make decisions regarding referrals to health care facilities based on what is in the best interest of patients without being induced by payments from hospitals competing for their business," said Bill Killian, U.S. Attorney for the Eastern District of Tennessee.
"Improper business deals between hospitals and physicians jeopardize both patient care and federal program dollars," said Daniel R. Levinson, Inspector General of the Department of Health and Human Services. "Our investigators continue to work shoulder to shoulder with other law enforcement authorities to stop schemes that imperil scarce health care resources."
Categories: Audits and Billing Issues, Fraud and Abuse, Hospitals, Medicare


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HCMP Update: Effects of COVID-19 on Leases in Washington
March 30, 2020 By HCMP Law Offices
Click here for a printable version.
The current global pandemic of the coronavirus disease (COVID-19) is affecting many aspects of daily life and business. Circumstances and guidance surrounding the spread of the disease change day by day, and governments are rapidly instituting emergency protocols, orders, and restrictions across industries in an attempt to support their citizens and economies.
As part of their emergency measures, jurisdictions throughout the state of Washington are instituting moratoriums on tenant evictions. This memorandum summarizes the current state of these moratoriums and analyzes their impact on landlords.
II. General Background
The first confirmed case of COVID-19 in the United States was announced by the state of Washington on January 21, 2020. The first deaths were announced in late February. As of March 2020, Washington has one of the highest reported numbers of confirmed cases in the country.
In response to the outbreak, the state of Washington and many of its local jurisdictions have begun issuing emergency orders relating to the administration of leases. Washington landlord and tenant law is codified in Title 59 of the Revised Code of Washington ("RCW"); the residential landlord-tenant act is codified in RCW 59.18 et seq.
III. Eviction Moratoriums by Jurisdiction 1
A concise summary of the key elements of the moratoriums currently in effect can be found in Section IV. A more detailed explanation of the individual policies follows.
a. The State of Washington
On March 18, 2020, the governor issued a proclamation "prohibit[ing] the following activities related to residential evictions by all residential landlords operating residential rental property in Washington State":
Residential landlords are prohibited from serving a notice of unlawful detainer for default payment of rent related to such property under RCW 59.12.030(3).
Residential landlords are prohibited from issuing a 20-day notice for unlawful detainer related to such property under RCW 59.12.030(2), unless the landlord attaches an affidavit attesting that the action is believed necessary to ensure the health and safety of the tenant or other individuals.
Residential landlords are prohibited from initiating judicial action seeking a writ of restitution involving a dwelling unit if the alleged basis for the writ is the failure of the tenant or tenants to timely pay rent. This prohibition includes, but is not limited to, an action under Chapters 59.12 or RCW 59.18 RCW
Local law enforcement is prohibited from serving or otherwise acting on eviction orders that are issued solely for default payment of rent related to such property. Nothing in this Proclamation is intended to prohibit local law enforcement from acting on orders of eviction issued for other reasons, including but not limited to waste, nuisance or commission of a crime on the premises.
The moratorium is initially in place for 30 days, concluding Friday, April 17, 2020.
The proclamation warns that landlords in violation of the order "may be subject to criminal penalties pursuant to RCW 43.06.220(5)," which is the statute proscribing the governor's powers in a state of emergency. Pursuant to this subsection, "[a]ny person willfully violating any provision of an order issued by the governor under this section is guilty of a gross misdemeanor."
In essence, the moratorium takes away a landlord's enforcement rights under RCW 59.12 (Unlawful Detainer) and RCW 59.18 (Residential Landlord Tenant Act) to remove residential tenants for the nonpayment of rent. Removal for valid criteria unrelated to the payment of rent remains an option, and the calculation of default fees is not addressed in the proclamation.
b. The City of Seattle – Residential
On March 14, 2020, the mayor issued an emergency order placing a temporary moratorium on residential evictions. On March 16, 2020, the City Council modified, expanded, and approved of the order. Pursuant to the expanded order:
A. Effective immediately, a moratorium on residential evictions is hereby ordered until the earlier of the termination of the civil emergency declared in the Proclamation of Civil Emergency dated March 3, 2020 or 60 days from the effective date of this Emergency Order. The decision to extend the moratorium shall be evaluated and determined by the Mayor based on public health necessity;
B. A residential landlord shall not initiate an unlawful detainer action, issue a notice of termination, or otherwise act on any termination notice, including any action or notice related to a rental agreement that has expired or will expire during the effective date of this Emergency Order, unless the unlawful detainer action or action on a termination notice is due to actions by the tenant constituting an imminent threat to the health or safety of neighbors, the landlord, or the tenant's or landlord's household members. Further, no late fees or other charges due to late payment of rent shall accrue during the moratorium; and
C. It shall be a defense to any eviction action that the eviction of the tenant will occur during the moratorium, unless the eviction action is due to actions by the tenant constituting an imminent threat to the health or safety of neighbors, the landlord, or the tenant's or landlord's household members. For any pending eviction action, regardless if the tenant has appeared, a court may grant a continuance for a future court date in order for the matter to heard at a time after the moratorium is terminated; and
D. Effective immediately, the Sheriff of King County is requested to cease execution of eviction orders during the moratorium.
The moratorium is initially in place for 60 days, concluding Friday, May 15, 2020.
This moratorium is more expansive that the one imposed over the state for three general reasons: (1) the 60-day effective period, (2) banning fees for late payment of rent during the moratorium, and (3) rather than limiting evictions for solely rent-related reasons, all residential evictions other than those related to tenant actions imminently threatening the health or safety of others are prohibited.
In sum, the moratorium takes away a landlord's enforcement rights under RCW 59.12 (Unlawful Detainer) and RCW 59.18 (Residential Landlord Tenant Act) to remove residential tenants unless the tenant is an imminent threat to health or safety, and it prohibits late fees for non-payment of rent.
c. The City of Seattle – Commercial
On March 17, 2020, the mayor issued an emergency order placing a temporary moratorium on small business evictions. Pursuant to the order:
A. Effective immediately, a moratorium on small business and nonprofit tenant evictions for non-payment of rent or due to the expiration of the lease's term are hereby ordered until the earlier of the termination of the civil emergency declared in the Proclamation of Civil Emergency dated March 3, 2020 or 60 days from the effective date of this Emergency Order. The decision to extend the moratorium shall be evaluated and determined by the Mayor based on public health necessity;
B. For purposes of this moratorium, "small business" shall have the same meaning as in RCW 19.85.020(3): "Small business' means any business entity, including a sole proprietorship, corporation, partnership, or other legal entity, that is owned and operated independently from all other business, and that has fifty or fewer employees" per establishment or premises;
C. For purposes of this moratorium, a "nonprofit" shall mean an entity that: (1) is a "not for profit corporation" or "nonprofit corporation" under RCW 24.03.005(16) or is designated a "public benefit not for profit corporation" or "public benefit nonprofit corporation" under RCW 24.03.490; (2) holds tax-exempt status under 26 U.S.C. § 501(c)(3);
The moratorium is effective for 60 days from entry of the order, or until May 16, 2020.
This moratorium bans late fees, interest, or other charges due to late payment of rent during the moratorium, and requires owners to "endeavor to enter into a payment plan, or other workout agreement to assist a distressed small business or nonprofit in rent relief, including but not limited to the deferred payment of rent, discount to rent, or other strategies to address the economic disruption caused by the COVID-19 civil emergency."
d. The City of Everett
On March 17, 2020, the mayor issued an emergency order placing a temporary moratorium on residential evictions. Pursuant to the order:
A. Effective immediately, a moratorium on residential evictions for non-payment is hereby ordered until the earlier of the termination of the Civil Emergency declared in the Proclamation of Civil Emergency dated March 4, 2020, or 30 days from the effective date of this Emergency Order. The decision to extend the moratorium shall be evaluated and determined by the Mayor based on public health necessity.
B. An owner of a housing unit shall not issue a notice of termination or initiate an eviction action for non-payment of rent or otherwise act on a termination notice for non-payment of rent during this moratorium. Further, no late fees or other charges due to late payment of rent shall accrue during the moratorium.
C. For any pending eviction action for the non-payment of rent, it shall be a defense to any eviction action that the eviction of the tenant would occur during the moratorium. Given the public health emergency and public safety issues, a court may grant a continuance for a future hearing date for the eviction action to be heard after the moratorium.
D. In any action seeking a writ of restitution involving a dwelling unit where the alleged basis for the writ is the failure of the tenant or tenants to timely pay rent, it shall be a defense to the action that writ would be executed before the termination of the Civil Emergency declared by the Mayor on March 4, 2020. Given the public health emergency and public safety issues, a court may grant a continuance for a future hearing date for the eviction action to be heard after the moratorium.
The moratorium is initially in place for 30 days, concluding Thursday, April 16, 2020.
Functionally, the moratorium provides the same limitations as that issued by the state of Washington.
e. The City of Burien
On March 23, 2020 the city council passed Resolution No. 426 to approve a moratorium on residential evictions. The resolution supports the state's moratorium on residential evictions, and adds the following requirement:
A landlord or owner of a residential housing unit shall not require payment o flate fees or charges due to delinquent payment of rent during this temporary moratorium. All residential tenant late fees and charges shall be waived during the Governor's temporary moratorium on evictions.
Therefore, the moratorium expands upon the one imposed over the state banning fees for late payment of rent during the moratorium.
f. The City of Renton
On March 23, 2020, the Renton City Council passed Resolution No. 4404 supporting the state's moratorium on residential evictions. At this time, the City of Renton has not expanded upon the state's moratorium.
g. The City of Auburn
On March 23, 2020, the Auburn City Council passed Resolution No. 5506 to approve a moratorium on residential evictions. The resolution supports the state's moratorium on residential evictions, and adds the following requirement:
The City Council ratifies the Mayor's Emergency Proclamation 2020-03 establishing a 30-day temporary moratorium restricting a landlord or owner of a residential housing unit form requiring payment of late fees or charges due to delinquent payment of rent. All residential tenant late fees and charges shall be waived during the temporary moratorium.
Therefore, the moratorium expands upon the one imposed over the state banning fees for late payment of residential rent during the moratorium.
Also on March 23, 2020, the Auburn City Council passed Resolution No. 5508 to approve a moratorium on commercial evictions. The resolution states:
No landlord or commercial property owner shall evict a commercial tenant in the City of Auburn during this local emergency period if the tenant provides one week notice to the landlord or commercial property owner before payment is due that the tenant is unable to pay rent dur to circumstances related to the COVID-19 pandemic. Within one week of the tenant's notice, the tenant must provide the landlord or commercial property owner with documentation supporting the tenant's inability to pay rent due to circumstances related to the COVID-19 pandemic.
These circumstances include loss of business income due to a COVID-19 related workplace closure, child care expenditures due to school closures, health care expenses related to being ill with COVID-19 or caring for a member of the tenant's household who is ill with COVID-19, or reasonable expenditures that stem from government-ordered emergency measures.
Thus, so long as the tenant provides notice and documentation of hardship, commercial evictions are temporarily prohibited in the Auburn.
h. The Cities of Bellevue, Des Moines, Federal Way, and Lynnwood
At this time, these cities have not implemented any independent emergency orders affecting evictions. If you lease property in a city in Washington not included in this memorandum, please reach out to inquire about any additional local restrictions.
i. King County 2
On March 17, 2020, the King County Sheriff's office announced that effective immediately they are "temporarily suspending the service and enforcement of evictions until further notice." The language of the Sheriff's letter appears to include residential and commercial evictions, and "will remain in effect" until revoked.
In response, the King County Superior Court has stayed residential eviction cases until April 17, 2020, and stayed small business and nonprofit evictions in the City of Seattle until May 3, 2020. The court's emergency order reads:
. . . Therefore all hearings, motions, and trials on residential eviction cases are stayed until April 17, 2020. For the City of Seattle all hearings on small business and nonprofit evictions are stayed until May 3, 2020.
This order lists no exceptions for emergency eviction or unlawful detainer proceedings, so we recommend seeking the advice of an attorney should an emergency situation present itself.
j. Snohomish County 3
On March 19, 2020 the Snohomish County Sheriff's office announced that effective immediately they "will not proceed with any residential evictions filed solely on the basis of non-payment of rent." The proclamation states that it does not cover "commercial properties or evictions resulting from criminal behavior, nuisance, or other reasons." In response, the Snohomish County Superior Court issued an emergency order confirming the Sheriff's action. This moratorium remains in effect until April 17, 2020.
IV. Impact and Recommendations
At this time, due to significant overlap between these rapidly emerging policies, there are five key geographic areas to consider in administrating leases: (1) the state of Washington generally, (2) King County, (3) the city of Seattle, (4) the city of Burien, and (5) the city of Auburn.
The moratorium covering the state of Washington prohibits residential evictions based on the nonpayment of rent for at least 30 days. A landlord may still remove a tenant for valid criteria unrelated to the payment of rent, including but not limited to waste, nuisance, or commission of a crime on the premises. A landlord's willful violation of the order may constitute a gross misdemeanor.
Practically, we advise that you take the following actions in Washington:
Stop all action on any ongoing default and eviction actions related to payment of rent;
Do not initiate new default or eviction actions related to payment of rent; and,
Set up a chain of command to evaluate and give permission for any and all default or eviction actions not related to the payment of rent in this time period.
The King County Superior Court has stayed residential eviction cases until April 17, 2020, and stayed small business and nonprofit evictions in the City of Seattle until May 3, 2020. The King County Sheriff's office has temporarily suspended "the service and enforcement of evictions until further notice." The language of the Sheriff's letter appears to include residential and commercial evictions, and "will remain in effect" until revoked.
In King County, we advise that you take the following additional actions:
Stop all action on any ongoing residential, small business, or nonprofit default and eviction actions for any reason;
Do not initiate new residential, small business, or nonprofit default or eviction actions for any reason;
Contact your lawyer in King County to discuss any and all default or eviction actions not related to the payment of rent in this time period and your possible options for emergency enforcement.
The city of Seattle has issued a more expansive 60-day moratorium on residential evictions, barring landlords from removing tenants for any reason unless the tenant is an imminent threat to health or safety. The city has further prohibited the accumulation of late fees for non-payment of rent during the moratorium.
The city of Seattle has also issued a 60-day moratorium on commercial evictions for small businesses and non-profits. This moratorium bans late fees, interest, or other charges due to late payment of rent during the moratorium, and requires owners to "endeavor to enter into a payment plan, or other workout agreement to assist a distressed small business or nonprofit in rent relief, including but not limited to the deferred payment of rent, discount to rent, or other strategies to address the economic disruption caused by the COVID-19 civil emergency."
And, we advise that you take the following actions in Seattle, specifically:
Stop all action on any ongoing default and eviction actions for any reason;
Do not initiate new default or eviction actions for any reason;
Stop calculating, and remove from accounting, any late fees or other charges incurred due to late payment of rent accruing during the moratorium in the city of Seattle;
Set up a chain of command to address and implement payment plans or workout agreements for any tenant that constitutes a small business or non-profit and fails to make rent payments in this time period; and,
Set up a chain of command to evaluate and give permission for any and all default or eviction actions relating to an imminent threat to health or safety in this time period.
The city of Burien has added to the state's residential eviction moratorium a requirement that landlords not charge late fees during the moratorium. Therefore, we advise that you take the following actions in Burien, specifically:
Stop calculating, and remove from accounting, any late fees or other charges incurred due to late payment of residential rent accruing during the moratorium.
The city of Auburn has also added to the state's residential eviction moratorium a requirement that landlords not charge late fees during the moratorium. Auburn has also placed a temporary moratorium on commercial evictions. Therefore, we advise that you take the following actions in Auburn, specifically:
Stop calculating, and remove from accounting, any late fees or other charges incurred due to late payment of residential rent accruing during the moratorium;
Set up a chain of command to address and implement documentation of hardships for commercial tenants who cannot pay rent in this time period.
The state of Washington and several of its cities and counties are rolling out emergency measures to combat the effect of the COVID-19 pandemic. In particular, several moratoriums on tenant evictions and late fees have been implemented or are being considered. This is a rapidly evolving situation, and landlords in the state of Washington must keep up with ever-changing requirements for the administration of leases.
Please do not hesitate to reach out to us with questions, and we hope that this memorandum will be of help in your business pursuits in Washington.
1 Because circumstances are evolving, it is likely that additional moratoriums, or extensions of moratoriums, will arise after the date of this memorandum.
2 The cities of Seattle, Burien, Bellevue, Renton, Auburn, Des Moines, and Federal Way are in King County.
3 The cities of Everett and Lynnwood are in Snohomish County.
Contributors to this article:
Michelle Gail | michelle.gail@hcmp.com | 206.470.7660
Mallory Satre | mallory.satre@hcmp.com | 206.470.7616
Chris Addicott | chris.addicott@hcmp.com | 206.470.7649
NOTE: This memorandum was published on March 30, 2020. The status and requirements of these moratoriums change daily.
The information contained in this update is provided for informational purposes only. It is not business, legal, accounting, tax, financial, investment or other advice on any matter and may not be relied upon as such. Please contact your attorney to obtain advice regarding any particular legal matter.

redpajama_set_name: RedPajamaCommonCrawl


When Jennifer was diagnosed with breast cancer in 2013 at the age of 33, she began to reevaluate her life's direction. Although seemingly content with most aspects of her life, Jennifer realized that she was just going through the motions. She found herself surrounded by poor relationships and stressful situations and spent little time involved in activities that she actually enjoyed. Confronted with a compilation of behaviors which were leading her to a life of poor health and chronic disease, she began looking for ways to regain control of her overall well-being. Through her own extensive research, Jennifer recognized that she had been creating the ideal environment within her body to promote cancer growth and she immediately set out to make substantial changes.
Jennifer will share her journey through her most radical change, from a Standard American Diet (SAD) to a Whole Foods Plant Based Diet (WFPBD), and describe the challenges she encountered along the way. With goals initially aimed at reducing her refined sugar and carbohydrate intake while adding nutrient dense foods to her diet, she had no serious intent of ever becoming a vegan. She will discuss the results she initially experienced and what motivated her to further refine her goals to include not only the removal of all animal products from her diet, but eliminating all highly processed foods and genetically modified organisms as well. Jennifer will share how these changes impacted her physical health as well as her mental and social well-being resulting in a level of wellness that she never could have envisioned. Delighted with her results, an interest in nutritional information was generated and she embarked on the task of enlightening others on how they too could make strides to improving their health.
Jennifer has achieved certification in plant-based nutrition from the T. Colin Campbell Foundation at eCornell University, and currently teaches health and science at the New Hampshire State Prison for Men. Jennifer's passion lies in informing and educating adults about their role in preventing chronic disease. She seeks to assist others in identifying their own nutrient deficiencies along with providing knowledge regarding the role nutrients play in the body, and how they can best be attained. Jennifer assists individuals in their shift to a more WFPBD and firmly believes that through proper nutritional education, and the implementation of adequate nutrient intake acquired through a WFPBD, the risk of acquiring chronic diseases diminish. Jennifer stresses that regardless the condition you may find yourself in, it is never too late to improve your diet, thereby increasing your level of wellness, while reversing the damaging effects of the Standard American Diet.
Transitioning to eating plant-based involves more than just eating great food, to be successful, it takes a mindful awareness.
Understanding our motivations, the social norms that surround us, and then creating our intention are some of the key aspects that separate us from trying to eat vegan on a whim to long-term success.
Regardless of your reason for exploring vegan and plant-based food Mark Dillon will show you how to touch in to your core motivation.
Mark Dillon has been vegan for 22 years. He hosts the popular podcast Mindful Vegan where he interviews some of today's top experts in the vegan world as well as sharing his insights on how we can all bring vegan food into our life.
As the founder of Explore Raw™ Stephanie creates recipes, runs workshops and classes at her Cookery School in London and hosts life changing raw food events and retreats in Portugal. Stephanie launched the world's very first vegan raw food bootcamp in 2013 and continues to run annual Raw Juice Camp events today.
Recently appearing in various media such as The Guardian, The Sunday Times, Vogue, Porter, Vegan Food & Living magazine and most recently on TV on Channel 4's 'What Britain Bought in 2015 with retail guru Mary Portas' Stephanie hopes to help bring plant based living and Spiralizing to a more mainstream audience.
Stephanie's recipes focus on creative dishes, constructing transition recipes that make meal times exciting, simple, nourishing and fun! Her dishes often focus on activated living foods and Stephanie lives her bring your food to life! strapline by living and teaching the principles and importance of enzyme rich, activated living ingredients.
Author of the plant based recipe book Spiralize! and 6 online ebooks, Stephanie is an accomplished chef, teacher and a vibrant energy in the clean-living movement.
Stephanie's book Spiralize! (Interlink Books) can be purchased from Amazon and all good bookstores. Stephanie will bring a small number of signed books along on the day for sale. There may even be a few giveaways!
Come drum with us at NHVegFest 2016 to elevate your mood and build up your immune system! We'll start by learning some basic rhythms, then play a few songs and drum circle games. Please bring your non-animal skin drums or try out one of ours – we'll have extras available. All ages and experience levels welcome keeping in mind drummers under 14 must be accompanied by an adult. You are welcome to store your drums in the music area while you walk around NH VegFest 2016.
Chef Mary Lawrence will share tips and strategies to help you overcome any obstacle. She will address common concerns about where to get your protein, calcium and iron on a vegan diet, how to make food appealing to friends and family, what pantry staples are essential for every vegan household, and how to save time, money and your sanity in the kitchen.
Mary Lawrence is executive chef and owner of Well on Wheels, Connecticut's premier vegan personal chef service. She is a frequent guest on radio and TV, including WNPR's "The Colin McEnroe Show," WWLP-TV's "Mass Appeal," and WTNH-TV's "CT Style." Mary has a certificate in plant-based nutrition from the T. Colin Campbell Foundation at eCornell University and has taught at the Connecticut Culinary Institute and numerous programs and conferences throughout the U.S. Her recipes have been included in the Best of Vegan Cooking (2009) by Priscilla Feral and VegCooking.com. Her most recent cookbook, Easy Peasy Vegan Eats (2014), is a guide for busy people making the vegan transition. She is also a writer and food blogger, and her culinary creations can be followed on the blog, The Traveling Vegan Chef (http://wellonwheels.blogspot.com).
Veganism is mostly associated with diet. But there are many other things that have animal products other than just food. Cayla Mackey, co-founder of two vegetarian businesses and the largest online curated collection of animal-free products, explains how vegetarianism goes beyond just food. She has worked with 200+ companies that cruelty-free products, and researched thousands of items to determine their ethics. Learn how the car you drive, the electronics you use, and the house you live in can be part of your path to a cruelty-free life.
What is the difference between eating meat and wearing leather? Are vegetarians allowed to wear wool or down? What about silk, or beeswax? Now more than ever before, ethical fashion is enabling vegetarians to wear their ethics on their sleeves. Advances in animal-free technologies, synthetic materials, and cruelty-free products are changing how people buy. This talk will arm you with a better understanding of your options as an ethical consumer, and leave you empowered to vote every day with your wallet for a cruelty-free world.
In "Fermentation 101" Andy will share his techniques to help get you started fermenting.


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Read all our latest news, grant and stories of change articles and stay up to date on our events
We are hiring - Fundraising Advisor (Consultancy)
Type of Contract: Consultancy contract
Contracted Hours: 8 days per month
Length of Contract: March – June 2019 with the possibility of a 2-month extension
Location: Accra, Ghana
Closing Date: 27th February 2019
Planned Interview Date: Week commencing 4th March 2019
STAR Ghana Foundation is an independent Ghanaian organisation which creates platforms for ordinary people, particularly the most marginalised in society, to become active citizens who demand positive change in their lives and communities. Our vision is to see a well-informed and active Ghanaian citizenry able to contribute to transformational change that advances democracy, accountability and social inclusion. We want to increase the effectiveness of citizens and civil society to achieve an equitable, inclusive society by convening inclusive dialogue and collaboration; catalysing active citizenship and collective action; coordinating and supporting strategic partnerships; and facilitating continuous learning to fuel wider scale change.
The Fundraising Advisor is responsible for coordinating and driving the implementation of the Foundation's fundraising action plan and Ethical Fundraising and Investment Policy. S/he will play the lead role in laying the foundation and leverage relationships for a diversified funding base and a sustainable income stream for the STAR Ghana Foundation. This will include developing, packaging and marketing a range of high-quality STAR Ghana Foundation products and coordinating all fundraising approaches and submissions to prospective donors and partners.
The role will work with the STAR Ghana Foundation management, its Governing Council and the STAR-Ghana programme Consortium to refine and advance the Foundation's fundraising targets. S/he will undertake detailed scoping on priority foundations, grant-makers, official donors, business funders and individual givers and provide active scanning, analysis and follow-up on new funding and resourcing opportunities for the Foundation.
The role in collaboration with the Foundation's Fundraising Committee will coordinate and support the Foundation's networking and visibility with African and international peer networks related to philanthropy, foundations and grant-making. S/he will coordinate with the Foundation's communications team to align fundraising efforts with the branding and positioning strategy, review and improve the fundraising functionality of the Foundation's website and social media platforms.
The successful applicant will have a Master's degree in a relevant field; combined with a minimum of 10 years of relevant experience. You should have substantial experience in fundraising, resource mobilisation, public relations, marketing and/or business development at a managerial level. You need to have proven high standards of integrity and accountability, with a demonstrated commitment to the values and goals of the Foundation. You are experienced with donor approval, reporting and accountability requirements and managing donor and fundraising data, information and records in an efficient, accountable way.
You need to have strong experience and skills in programme design, proposal development, budgeting, project reporting, and donor/client management and the ability to manage and inspire patrons, champions, volunteers, partners and Governing Council members in their contributions to fundraising efforts.
You have excellent written, verbal and presentational skills to communicate complex ideas to a range of audiences. You have demonstrable public speaking, partnership-building, relationship management, advocacy, strategic thinking and analytical skills. Relevant experience in the civil society and development sectors. Specialist experience in resource mobilisation from business and CSR funders, aid donors, private foundations and/or individual givers.
Interested applicants should send their CV and cover letter to: This email address is being protected from spambots. You need JavaScript enabled to view it. not later than 17:00 GMT, 27th February 2019
2nd Annual Strategic Learning Event held in Accra
After two years of programme implementation, STAR-Ghana Phase 2 has made substantial progress in the rollout of key thematic grant calls, the '3C&L' approach and Learning Framework (Communities of Practice and Learning - CoPL). It has successfully launched the STAR-Ghana Foundation in November 2018.
Hajia Hamdiya Ismaila, member of the Governing Council of STAR Ghana Foundation, officially opened the event
The programme continues to support a wide range of strategic and small grant partner initiatives across the country – engaging with national and district level actors to strengthen civil society engagement with government for increased transparency, accountability and responsiveness.
A cross-section of participants at the event
As experience accumulates from collaboration with grant partners, the establishment of the new Foundation, and application of the 3Cs&L mission (convening, coordination, catalyst and learning), this event created space for the STAR-Ghana team – together with Programme Consortium Partners and other key stakeholders to pause once again for strategic reflection to harvest and apply lessons from practice.
Eunice Agbenyadzi, GESI & CB Manager, sharing Learning from the GESI work-stream with some participants at the event
The three-day workshop on the theme: "Lessons for Active Citizenship", was attended by over 70 participants.
Media Coalition Storms Regions To Whip up Support For RTI Bill
Participants in Cape Coast
The Media Coalition on the Right To Information (RTI) Bill, represented by Parliamentary News Africa (PNAfrica), and with funding from Star Ghana Foundation, is currently on a tour of five regions to gunner support for the passage of the bill.
The tour dubbed, Regional RTI Media Sensitization Workshops started on Monday in the Volta and Ashanti regions, with Brong Ahafo and Central regions following suite on Tuesday and Wednesday respectively. The Northern Region climaxing it on Thursday.
The purpose of the sensitization workshops is to educate radio programme hosts, editors, journalists and producers of morning shows on the RTI Bill to enable them adequately inform and empower their listeners, viewers and readers. This is expected to raise enough nationwide public awareness about the bill and court regional public support to pressurize Members of Parliament to pass the bill into a credible law.
Resource Persons from the Media Coalition on RTI took participants through the journey of the RTI Bill in parliament, the memorandum to the bill, and the success story of the media coalition.
At the end of the regional RTI media sensitization workshops, all the beneficial regions are expected to come out with their own advocacy campaign strategy that would result in the passage of the bill.
The Right to Information is a fundamental human right guaranteed by the Constitution of the Republic of Ghana and recognized as a right by international conventions on human rights. Ghana's choice of democratic governance requires an active participation by all in the governance process, thereby making the right to information particularly relevant. It is only when those who are to participate in governance are well informed that they can contribute meaningfully.
However, due to the lack of political will, for about two decades now, successive governments have failed to ensure the passage of an RTI law that will give meaning to this constitutional right. Several versions of an RTI Bill have been presented to Parliament since 2010, yet the House of Representatives have failed to take the Bills through all the stages of the law-making process. The latest version of the Bill, which was laid in May 2018, is currently at the Consideration Stage in Parliament.
Volta Region Participants
STAR Ghana Foundation celebrates Prof. Henrietta Mensa-Bonsu, Governing Council Member
On Thursday 17th January 2019, the Ghana Academy of Arts and Sciences inducted into office, the 37th Council to steer the affairs of the nation's premier learned body for a two-year term; from January 2019 to December 2020. The election of the new officers took place at the Academy's 199th General Meeting of Fellows in December 2018.
The President of the new Council is Prof. Henrietta Mensa-Bonsu, a Professor of Law, immediate past Director of the Legon Centre for International Affairs and Diplomacy; and former Deputy Special Representative (Rule of Law) to the United Nations Secretary General Ban Ki-moon, for Liberia.
Prof. Henrietta Mensa-Bonsu currently serves as a Governing Council Member of the STAR Ghana Foundation which is the highest decision-making body steering the affairs of the Foundation.
STAR Ghana Foundation and the Programme Management Team of STAR-Ghana, express our profound and heartfelt congratulations on your election as the President of the Ghana Academy of Arts and Sciences.
With Thanks to our Funders
Sign up to the STAR-Ghana newsletter to get the latest news and updates
No 12, Obenesu Crescent, East Cantonments, Accra-Ghana
info@star-ghana.org
© All Rights Reserved, STAR-Ghana. Inspired By DreamOval


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NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS| Volume 273, ISSUE 9, P5204-5210, February 27, 1998
PDF [223 KB]PDF [223 KB]
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Analysis of Strand Slippage in DNA Polymerase Expansions of CAG/CTG Triplet Repeats Associated with Neurodegenerative Disease*
John Petruska
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340
Michael J. Hartenstine
Myron F. Goodman
* This work was supported by National Institutes of Health Grants AG 11398 and GM 21422.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessDOI:https://doi.org/10.1074/jbc.273.9.5204
Previous ArticleUltraviolet Radiation-induced Ubiquitination and Proteasomal Degradation of the Large Subunit of RNA Polymerase II
Next ArticleGenomic Organization and Identification of an Enhancer Element Containing Binding Sites for Multiple Proteins in Rat Pituitary Tumor-transforming Gene*
Lengthy expansions of trinucleotide repeats are found in DNA of patients suffering severe neurodegenerative age-related diseases. Using a synthetic self-priming DNA, containing CAG and CTG repeats implicated in Huntington's disease and several other neurological disorders, we measure the equilibrium distribution of hairpin folding and generate triplet repeat expansions by polymerase-catalyzed extensions of the hairpin folds. Expansions occur by slippage in steps of two CAG triplets when the self-priming sequence (CTG)16(CAG)4 is extended with proofreading-defective Klenow fragment (KF exo−) fromEscherichia coli DNA polymerase I. Slippage by two triplets is 20 times more frequent than by one triplet, in accordance with our finding that hairpin loops with even numbers of triplets are 1–2 kcal/mol more stable than their odd-numbered counterparts. By measuring triplet repeat expansions as they evolve over time, individual rate constants for expansion from 4 to 18 CAG repeats are obtained. An empirical expression is derived from the data, enabling the prediction of slippage rates from the ratio of hairpin CTG/CTG interactions to CAG/CTG interactions. Slippage is initiated internally in the hairpin folds in preference to melting inward from the 3′ terminus. The same triplet expansions are obtained using proofreading-proficient KF exo+, provided 10–100-fold higher dNTP concentrations are present to counteract the effect of 3′-exonucleolytic proofreading.
The human genome has an abundance of simple sequence repetitions that are unstable and tend to expand in large numbers in some genetic loci. A prime example is the CAG/CTG class of triplet repeats whose large expansions occur in genes associated with Huntington's disease and six other neurological disorders (
Ashley Jr., C.T.
Warren S.T.
Annu. Rev. Genet. 1995; 29: 703-728
). Such expansions represent a novel form of mutation whose cause is unknown. An attractive possibility under investigation is primer/template slippage during DNA replication or repair of tandemly repeated sequences (
Sinden R.R.
Wells R.D.
Curr. Opin. Biotechnol. 1992; 3: 612-622
J. Biol. Chem. 1996; 271: 2875-2878
Richards R.I.
Sutherland G.R.
Nat. Genet. 1994; 6: 114-116
Schlotterer C.
Tautz D.
Nucleic Acids Res. 1992; 20: 211-215
Behn-Krappa A.
Doerfler W.
Hum. Mutat. 1994; 3: 19-24
Ji J.
Clegg N.J.
Peterson K.R.
Jackson A.L.
Laird C.D.
Loeb L.A.
Nucleic Acids Res. 1996; 24: 2835-2840
Ohshima K.
J. Biol. Chem. 1997; 272: 16798-16806
Strand slippage can occur when there is local strand separation in DNA regions containing tandem repetitions (
Levinson G.
Gutman G.A.
Mol. Biol. Evol. 1987; 4: 203-221
). For example, in a region of CAG/CTG repeats, local separation creates single-strand loops of CAGs and CTGs whose repetitive character may allow the two strands to be displaced (i.e. slipped) by an integral number of triplets. Such slippage in the presence of DNA polymerases can result in the addition of integral numbers of triplets to give triplet repeat expansions (
). Large loops that form stable hairpin structures are expected to increase the probability of slippage and expansion (
Darlow J.M.
Leach D.R.F.
Genetics. 1995; 141: 825-832
Kang S.
Shimizu M.
Amirhaeri S.
Gacy A.M.
Goellner G.
Juranic N.
Macura S.
McMurray C.T.
Cell. 1995; 81: 533-540
Mitas M.
Yi A.
Dill J.
Kamp T.J.
Chambers E.J.
Haworth I.S.
Petruska J.
Arnheim N.
Goodman M.F.
We (
) and others (
Smith G.K.
Jie J.
Fox G.E.
Gao X.
) have shown that single strands of CTG repeats form stable hairpin structures as a result of base pairing between antiparallel CTGs, yielding G·C and C·G base pairs alternating with T·T mispairs. Similar hairpins also form in strands of CAG repeats but are not as stable (
Yu A.
), probably because A·A mispairs are bulkier and more destabilizing than T·T (
Aboul-ela F.
Koh D.
Tinoco I.J.
Martin F.H.
). Our thermal denaturation studies of strand folding (
) indicated that hairpin stability increases more slowly than expected with increasing numbers of repeats. A plausible explanation is that strands with more repeating triplets have more degrees of freedom and tend to form several shorter hairpin loops instead of one long one. For example, a strand with 10 triplet repeats may fold into a single hairpin structure, whereas a longer strand with 30 repeats may form a more complex structure with two or three hairpin folds of similar size (
Evidence of strand slippage in CAG/CTG repeat regions during DNA polymerization has been obtained using various gel-based primer extension assays (
). Our assay differs from earlier ones mainly in the priming event. We engineered a DNA oligonucleotide of triplet repeats so that it is primed by intramolecular folding, rather than by intermolecular association. Being independent of concentration, the self-priming remains efficient at low DNA concentrations, unlike intermolecular priming that requires higher DNA concentrations. By using low concentrations, we avoid uncertainties arising from multimeric associations between DNA strands. Also, our starting primer 3′-end is fully complementary to template repeats, instead of partially complementary as in the only previous study of self-priming with triplet repeats (
In the present study we use a self-priming strand containing a large number of CTGs relative to CAGs, to measure the influence of CTG/CTG interactions on strand slippage in polymerase-catalyzed expansions of correctly paired (CAG/CTG) repetitions. This approach provides for the first time an opportunity to measure slippage and expansion rates as a function of the number of interacting triplets, for DNA polymerases with and without proofreading 3′-exonuclease activity.
Escherichia coli DNA polymerase I Klenow fragment (KF)
The abbreviations used are: KF, Klenow fragment;A, self-priming DNA sequence, 5′-(CTG)16(CAG)4-3′; An, initial hairpin fold of sequence A, having n CTG triplets at the 5′-end available as template for extending the primer 3′-end by n CAG triplets; An + (CAG)n, blunt-end hairpin formed by extendingAn by n CAG triplets with DNA polymerase.
mutant, exo− (D355A,E357A), devoid of proofreading 3′-exonuclease activity, was purified from overproducing strains (
Derbyshire V.
Freemont P.S.
Sanderson M.R.
Beese L.
Friedman J.M.
Joyce C.M.
Steitz T.A.
Science. 1988; 240: 199-201
). Normal proofreading-proficient KF polymerase (exo+) was obtained commercially (U. S. Biochemical Corp./Amersham Corp.).
Oligonucleotide Synthesis
The self-priming DNA 60-mer, (CTG)16(CAG)4, and 30-mer (CTG)6(CAG)4 were synthesized by an Applied Biosystems DNA/RNA synthesizer, using β-cyanoethyl phosphoramidites, and purified by denaturing polyacrylamide gel electrophoresis. The 90-mer DNA marker (CTG)16(CAG)14 was purchased from Operon Technologies Inc. and obtained lyophilized after high pressure liquid chromatography purification. Purified DNA samples were dialyzed extensively against a low ionic strength buffer (5 mm NaH2PO4, 5 mmNa2HPO4, 1 mm Na4EDTA, pH 7.0) and stored at −70 °C.
Melting Analysis
Thermal denaturation profiles were obtained for the 60- and 30-mer DNAs at the same strand concentration (1.9 μm) in low ionic strength buffer, by measuring UV absorbance A260 versus temperature, while raising temperature from 25 to 85 °C at a constant rate of 2 °C/min.
End Labeling and Equilibration
Samples of the self-priming 60-mer used in polymerase reactions were 5′-end-labeled with32P, using [γ-32P]ATP and T4 polynucleotide kinase (U. S. Biochemical Corp./Amersham Corp.) in kinase buffer (50 mm Tris-HCl, pH 7.6, 10 mmMgCl2, and 10 mm 2-mercaptoethanol). Labeled samples at a strand concentration of 100 nm were heat-denatured at 100 °C for 5 min and allowed to renature by cooling at room temperature. The results obtained in polymerase reactions showed no dependence on the rate of cooling, as expected for intramolecular folding. The labeled strands were stored at 4 °C to avoid any potential intermolecular associations promoted by freezing (
Extension Reactions
Radiolabeled DNA at 10 nmstrand concentration was incubated 5 min at 37 °C in reaction buffer (50 mm Tris-HCl, pH 7.5, 10 mmMgCl2, 1 mm dithiothreitol, and 0.05 mg/ml bovine serum albumin) to allow equilibration of DNA structure. Typically, 60 μl of DNA solution was then micropipeted into 15 μl of enzyme + dNTP solution (same buffer) held in a microcentrifuge tube at 37 °C, at which point running time (t) for reaction was started. Final enzyme concentration was approximately 60 nm in each case; the dNTPs used (N = C, A, G) were in equimolar amounts ranging from 0.1 to 10 μm. At times indicated (t = 0.5 min, etc.) a 5-μl aliquot of reaction mixture was removed and quenched with 25 μl of 20 mm EDTA + 20 m formamide.
Denaturing Gel Electrophoresis
Reaction products were separated into bands according to product size, by denaturing gel electrophoresis at 2000 V on 12% polyacrylamide (40 cm × 40 cm × 0.2 mm) with 16 m formamide as denaturant, in TBE buffer (89 mm Tris borate, pH 8.3, 2 mmNa2EDTA). Gels dried on paper were scanned with Molecular Dynamics Storm 860 PhosphorImager, and band intensities were integrated by FragmeNT Analysis software. Each band intensity was evaluated as percent of total integrated band intensities in the corresponding lane.
Expansion Rate Analysis
The 60-mer sequence (A) forms hairpin loops (An) with even and odd numbers of bases in the hairpin bend (Fig. 1). In the presence of DNA polymerase and dNTPs, the loops are rapidly extended (in seconds) to blunt-ended forms, An + (CAG)n, seen as gel band intensities In, where n is the number of CAG triplets added (Fig. 3). The band intensities change gradually (in minutes) because of slippage and further expansion. Since bands with n = 0, 2, 4, etc. are much more intense than those with n = 1, 3, 5, etc., even-numbered loops are evidently more stable then odd-numbered, as suggested earlier (
). Thus slippage by two triplets and expansion by (CAG)2 is favored over slippage by one triplet and expansion by (CAG)1. To verify that added triplets have the sequence CAG, chain termination with dideoxy-NTPs is used. In separate reactions, a single dideoxy-NTP is added, at 100 × the corresponding dNTP concentration, to obtain termination atN = C, A, or G. In this way (data not shown) we confirm the positions of bands corresponding to addition of (CAG)n for both even and odd values of n.
Figure 1Possible hairpin folds of self-priming 60-mer DNA sequence (CTG)16(CAG)4. The even-numbered loops (A0,A2, etc.) and less stable odd-numbered loops (A1, A3, etc.) are extendable by even and odd numbers of CAG triplets, respectively. the solid dots indicate correct base pairs; the asterisk indicates the 5′-end radiolabeled with32P. The most stable loop is blunt-endedA0, having the most correct base pairs, namely 23. The loops indicated as much less stable have less than 4 CAGs correctly paired with complementary antiparallel CTGs. Upon reaction with DNA polymerase and dNTPs (N = C, A, G), the initial loops An are rapidly extended (in seconds) to blunt-end products, An + (CAG)n, seen as band intensities In (n = 0, 1, 2, etc.) by denaturing gel electrophoresis. With increasing reaction time, the changes in In indicate the rates of strand slippage in the blunt-ended hairpins and further expansion by addition of more CAG triplets.
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Figure 3Band patterns on denaturing gel obtained by extending self-priming 60-mer with DNA polymerase KF exo−. Extreme left and rightlanes contain the 90-mer DNA marker (M),32P(CTG)16(CAG)14. The second lane from the left shows the 60-mer32P(CTG)16(CAG)4 without polymerase. The other lanes show the 60-mer extended by the addition of CAG triplets with DNA polymerase + 0.2 μmdNTPs (N = C, A,G) at 37 °C, removing samples and quenching with EDTA at reaction times indicated.
Assuming band intensities change successively by 2-triplet slippage and expansion, so that In converts toIn+2 with rate constantkn, we evaluated rate constantsk0 for I0 →I2, k2 for I2 → I4, etc. (Table I). The evaluation is made by applying the differential equations describing the corresponding rates of intensity change with timet, namely dI0/dt = −k0 I0, dI2/dt =k0 I0−k2 I2, … , dIn/dt =kn−2 In−2−kn In. SinceI0 decays exponentially with t, the k0 value is obtained directly from the slope of the straight line fitted by least squares, logI0 = log I0(t→ 0) − k0 t, whereI0(t → 0) is the initialI0 found by extrapolating to t = 0. Then, using k0 and the value of I0 at each time point, we obtaink2 by a least squares fit to the rates of change of I2. Thus, after determiningk0 directly, we are able to determinek2 relative to k0,k4 relative to k2, etc.
Table IInitial amounts of hairpin folds in sequence A = (CTG)16(CAG)4 and corresponding slippage rate constants for 2-triplet expansions
Hairpin fold
Band intensity
Initial amount
Slippage expansion
Rate constant
% min−1
Even-numbered loops
A0 I0 = [A0] 61 I0 → I2 0.81 ± 0.02
A2 I2 = [A2 + (CAG)2] 7 I2 → I4 0.48 ± 0.02
A8 I8 = [A8 + (CAG)8] 6 I8 → I10 0.28 ± 0.02
A10 I10 = [A10 + (CAG)10] 7 I10 → I12 0.08 ± 0.03
A12 I12 = [A12 + (CAG)12] 1.5 I12 → I14 0.06 ± 0.03
A14 I14 = [A14 + (CAG)14] 0.5 I14 → I16 ND
Odd-numbered loops
A1 I1 = [A1 + (CAG)1] 2 I1 → I3 (0.7 ± 0.1)
Slippage rate constant k1 for I1 → I3, found along withk0′ = 0.04 ± 0.02 min−1 for I0 → I1 (slippage ofA0 by 1 triplet), by analyzing the changes in I1 versus t in accordance with dI1/dt =k0′I0 −k1I1.
A3 I3 = [A3 + (CAG)3] 1 I3 → I5 ND
A5 I5 = [A5 + (CAG)5] 0.5 I5 → I7 ND
1-a Slippage rate constant k1 for I1 → I3, found along withk0′ = 0.04 ± 0.02 min−1 for I0 → I1 (slippage ofA0 by 1 triplet), by analyzing the changes in I1 versus t in accordance with dI1/dt =k0′I0 −k1I1.
For comparison, we also evaluated a rate constant (k0′) for slippage by 1 triplet (I0 → I1), converting the even-numbered loop A0 to the odd-numbered loop A1 (Fig. 1). The assumption is thatI0 → I1 occurs with rate constant k0′, whereasI1 → I3 occurs with rate constant k1 for 2-triplet slippage. Then, applying the corresponding differential equation, dI1/dt =k0′I0 −k1 I1, we find thatk0′ (for 1-triplet slippage) is an order of magnitude less than k0 (for 2-triplet slippage), whereas k1 for 2-triplet slippage of an odd-numbered loop is consistent with k0 and k2 for 2-triplet slippage of even-numbered loops.
The self-priming sequence, A = (CTG)16(CAG)4, forms a series of hairpin loops (Fig. 1), to which integral numbers of CAG triplets can be added by polymerase-catalyzed reaction with appropriate dNTP substrates (N = C, A, G). In Fig. 1,A0 represents the most stable loop in the form of a blunt-ended hairpin; A1 corresponds to slippage of A0 by a single triplet;A2, slippage by two triplets; etc. Initially, there is an equilibrium distribution of loops reflecting their thermodynamic stability in the absence of polymerase. We verify that stable folds are formed by examining thermal denaturation profiles obtained by plotting UV absorbance (A260)versus temperature (Fig. 2).
Figure 2Comparison of thermal melting profiles for DNA 60-mer (CTG)16(CAG)4 and 30-mer (CTG)6(CAG)4. The heat denaturation curves and indicated melting temperatures in low ionic strength buffer, found by plotting UV absorbance A260 versus temperature, are shown at the same (1.9 μm) strand concentration.
When DNA polymerase and dNTPs are added, the original loops are extended to blunt ends in a matter of seconds, so that the rates at which blunt-end hairpins rearrange by slippage and expand by triplet additions can be accurately measured over a period of minutes. To establish the equilibrium amounts of individual hairpins and their extension rates, we analyzed polymerase-catalyzed reaction products separated into discrete bands by electrophoresis on a denaturing, formamide-polyacrylamide gel. The products are first analyzed as a function of reaction time (t) at physiologically low (0.1 to 1 μm) dNTP concentration, using a proofreading-deficient DNA polymerase, Klenow fragment exo−. The results shown (Fig. 3) are obtained with 0.2 μm each of the three dNTP substrates (N = C, A, G). This concentration appears high enough to obtain near-maximum velocity of extension with a minimum of artifacts such as terminal transferase activity, seen at higher [dNTP] as an increase in background "pause" bands (data not shown).
Thermal Denaturation Profiles
Upon heat denaturation (Fig. 2), the 60-mer (CTG)16(CAG)4 shows two sigmoidal transitions, with melting temperature Tm = 56 and 80 °C at low ionic strength. By comparison, the 30-mer (CTG)6(CAG)4, having 10 fewer CTG repeats, shows only the higher transition with Tm near 80 °C. The biphasic melting curve of the 60-mer is consistent with the kind of hairpin folding shown in Fig. 1, in which the 4 CAG triplets are correctly base-paired with 4 CTG repeats to give the more stable component melting at 80 °C, whereas the remaining 12 CTG triplets are more weakly paired to give the less stable component melting at 56 °C. The latter Tm value is close to that observed for hairpin folding of CTG triplets alone,e.g. (CTG)10 and (CTG)30, each withTm = 51 °C (
). However, the melting curve is unable to resolve the kinds of hairpin folds present. The various possible folds (Fig. 1) initially have the same strand length, but when extended with polymerase they acquire increasing lengths, which can be separated by electrophoresis on formamide-polyacrylamide gel.
Initial Hairpin Loops
As seen in Fig. 1, starting at the 5′-end marked by *, the 60-mer sequence A has a potential template of 16 CTG repeats followed by a primer of 4 CAG repeats. Upon hairpin folding, so that the CAGs align with CTGs in antiparallel fashion, the (CAG)4 primer can be correctly base-paired with any 4 successive antiparallel CTGs, shown as (GTC)4 in Fig. 1. The first loop, A0, is a blunt-ended structure with 3′- and 5′-ends juxtaposed. This is the most stable fold because it allows remaining CTGs to form the maximum number of base pairs with each other.
The even-numbered loops A2,A4, … , A10belong in the same group as A0, because they each have the same kind of 4-base hairpin bend and an even number of overhanging triplets. The odd-numbered loops A1,A3, … , A11belong in a separate group, because they each have a 3-base bend and an odd number of overhanging triplets. The even-numbered loops are expected to be somewhat more stable than the odd-numbered (
), because they each have one more base pair stabilizing the hairpin bend. For completeness, much less stable loops, A12 toA15, having fewer than 4 correctly paired CAG/CTG combinations, are also shown (Fig. 1).
Being blunt-ended, A0 is not suitable for extension by DNA polymerases until it rearranges by strand slippage into one of the other possible forms with an overhanging 5′-template, (GTC)n* (Fig. 1). These forms, An(n = 1, 2, etc.), are easily extended to blunt-end products, An + (CAG)n, by the addition of (CAG)n opposite (GTC)n*. The products are resolved as a series of bands on a denaturing gel, with band intensityIn proportional to the amount of product, (An + (CAG)n). The 5′-end (*) is radiolabeled with 32P to obtain quantitative band intensity measurements with a PhosphorImager.
Loop Resolution by Electrophoretic Analysis of Polymerase-catalyzed Extension Products
Using proofreading-deficient DNA polymerase KF exo−, we observed complete extension of initial loops within 0.5 min of reaction (Fig. 3). A 6-fold excess of polymerase is used to saturate all the self-primed loops originally present so that they can be completely extended to blunt ends in seconds, before appreciable primer/template slippage can occur. Also, we use a dNTP concentration low enough (0.2 μm) to avoid the formation of anomalous pause bands arising by base misinsertion or by blunt end addition of a single nucleotide (
Clark J.M.
In Fig. 3 it is clear that the most intense bands correspond to additions of even numbers of CAG triplets. The bands formed by adding 0, 2, 4, etc. triplets are much more intense than those formed by adding 1, 3, etc. triplets. This striking result shows that even-numbered loops (Fig. 1) are significantly more abundant,i.e. more stable, than odd-numbered, in agreement with earlier observations (
). The way the band intensitiesIn change with t, the time of reaction, indicates the rates at which the blunt-end loops, An+ (CAG)n, rearrange by slippage and expand by the addition of more CAGs.
Measurement of Expansion Rates for Primer/Template Slippage in Polymerase KF exo− Reactions
By examining the band patterns in Fig. 3, we find that as the original 60-mer band (0 triplets added) decays with t, each subsequent even-numbered band, representing addition of 2, 4, etc. CAG triplets, intensifies to a maximum before decaying. This is better illustrated in Fig. 4, where the band intensityIn, expressed as percent of total intensity in each lane, is shown plotted versus t for the first three even-numbered bands, n = 0, 2, and 4. Since odd-numbered bands (additions of 1, 3, etc. triplets) remain faint at all times, we see that slippage by two triplets is strongly favored over slippage by one triplet.
Figure 4Plot of band intensities versusreaction time for the first three main bands representing extensions by 0, 2, and 4 CAG triplets. Band intensity I0indicates the amount of the original blunt-ended loopA0 (Fig. 1), and I2 and I4 indicate the amounts of the extension products, A2 + (CAG)2 andA4 + (CAG)4, respectively. Each band intensity is expressed as percent of total integrated band intensities in the corresponding lane (Fig. 3), measured by PhosphorImager.
To describe how each band intensity In changes because of slippage by 2 triplets and subsequent expansion by (CAG)2, we use the kinetic scheme in Fig. 5. The rate constantskn with n = 0, 2, 4, etc. represent "macroscopic" rates for converting each even-numbered blunt-end loop (In) to the next (In+2) by 2-triplet slippage and expansion. For n = 0, the intensityI0 decreases exponentially with t(Fig. 4), and the exponential curve fitted by least squares yields the slippage rate constant, k0 = 0.81 min−1. By extrapolating to t = 0, we findI0(t → 0) = 61% as the initial amount of hairpin A0 (Table I). For n = 2, band intensity I2 rises rapidly until it reaches a maximum and then gradually declines (Fig. 4). By assumingk0 is the rate constant for convertingI0 to I2 by 2-triplet slippage, we obtain a good fit with the relationship, dI2/dt =k0 I0 −k2 I2, enabling us to determine k2 relative tok0. Also, by extrapolating to t= 0 we obtain I2(t → 0), the initial amount of hairpin A2 (Table I). Similarly, using dIn/dt =kn−2 In−2− kn In, we are able to evaluatekn relative to kn−2and by extrapolation to estimate the initial amounts of the even-numbered hairpins with n = 4–14, i.e. A4 to A14. In the case ofA14, the initial amount (0.5%) is barely above the minimum level of detection, and the rate constant for slippage is not determined (ND, Table I).
Figure 5Kinetic scheme used to evaluate rate constants for slippage and expansion by two triplets. The rate constants kn (n = 0, 2, 4, etc.) indicate the rate at which band intensity In is converted to In2 as a result of 2-triplet slippage and polymerase-catalyzed expansion by 2 CAG triplets.
Because odd-numbered loops give much less intense bands, comparable to faint pause bands in the background, we can only evaluate initial amounts for A1, A3, andA5 (Table I). In the case ofA1, there is sufficient intensity to estimate a rate constant k1 for 2-triplet slippage (I1 → I3), assumingA0 contributes to A1(I0 → I1) with rate constant k0′ for 1-triplet slippage. In this case, dI1/dt =k0′I0 −k1 I1, when applied to our data, yields k0′ = 0.04 ± 0.02 min−1 and k1 = 0.7 ± 0.1 min−1 (Table I, in brackets).
Relationship between Slippage Rate and Number of Interacting Triplets
In Table I (last column) we see that the slippage rate constant kn decreases with increasing number,n, of added CAGs. The rate constant is highest (0.8 min−1) for n = 0, the initial blunt-end hairpin A0 (Fig. 1), which has the highest number of CTG/CTG interactions relative to CAG/CTG interactions. As Fig. 1 shows, A0 has 4 strong CAG/CTG base pairing interactions and 5 weaker CTG/CTG interactions, so that (CTG/CTG)/(CAG/CTG) = 5/4. The longer blunt-end hairpins,An + (CAG)n, formed by adding nCAG triplets complementary to the template (GTC)n* (Fig. 1), gain n more CAG/CTG interactions while losing n/2 CTG/CTG interactions. Thus, while (CAG/CTG) = 4 + nincreases with n, both (CTG/CTG) = 5 − (n/2) and the ratio, (CTG/CTG)/(CAG/CTG) = (10 −n)/(8 + 2n), decrease with n.
The kn value (on a logarithmic scale) shows a positive correlation with (CTG/CTG) = 5 − n/2 and a negative correlation with (CAG/CTG) = 4 + n (Fig. 6 A). In each case, the correlation is nonlinear for even values of n from 0 to 8. However, when log kn is plotted against the ratio, (CTG/CTG)/(CAG/CTG) = (10 − n)/(8 + 2n), the points fall close to a straight line (Fig. 6 B). Furthermore, k1 obtained for 2-triplet slippage between odd-numbered loops (Table I, in brackets) also falls on this line, at the point corresponding to n = 1 (Fig. 6 B, open square).
Figure 6Semi-log plot of rate constant values as a function of interacting triplets. A, plot of logkn (n = 0, 2, … , 10)versus the corresponding number of triplet interactions, (CAG/CTG) = 4 + n (upper scale) or CTG/CTG = 5 − (n/2) (lower scale). B, plot of log kn versus the relative number of CTG/CTG interactions to CAG/CTG interactions, i.e.(CTG/CTG)/(CAG/CTG) = (10 − n)/(8 + 2n). The straight line is a least squares fit tok0, k2, … ,k10 (solid squares) found for 2-triplet slippage in even-numbered loops; the open squareis the lone (k1) value found for 2-triplet slippage in odd-numbered loops (Table I, brackets).
The equation of the line fitted by least squares (Fig. 6 B) is log10 kn = 0.38x − 0.56, where x = (CTG/CTG)/(CAG/CTG). This equation can also be written as kn = αe βx, where α = 0.28 min−1 and β = 0.87. We can interpret α as the slippage rate constant for CAG/CTG interactions alone and e βx as the factor arising from the influence of (CTG/CTG) on the slippage of (CAG/CTG). This factor appears to hold as long as slippage by 2 triplets does not change the character of the hairpin bend made by CTGs.
The even-numbered loops A0 toA10 (Fig. 1) have the same kind of 4-base bend made with two CTGs. However, in A12 the bend acquires a CAG in place of a CTG at the expense of a CAG/CTG base-pairing interaction, making it more difficult for A10 → A12 slippage to occur. As seen in Table I (last column), the slippage rate constant drops dramatically, from 0.28 min−1 for k8 (I8 →I10) to 0.08 min−1 for k10 (I10 →I12). This marked reduction in slippage rate is also evident in the accumulation of band intensityI10, corresponding to 10 CAG triplets added (Fig. 3). Subsequently, the slippage rate declines further to 0.06 min−1 for k12(I12 → I14) and to an even lower value for k14, undetermined (ND) in Table I.
Effect of Proofreading Activity on Expansion Rate
When KF exo− is replaced by normal proofreading-proficient Klenow fragment, the 3′-exonuclease activity of proofreading markedly inhibits expansion at low dNTP concentrations (Fig. 7). At 0.2 μm dTNP, band intensities similar to those found initially with KF exo−are observed in the 1st min of reaction, but expansion by slippage is greatly reduced because of 3′-end degradation with increasing time (Fig. 7). As dNTP concentration is increased to counteract the degradation, the band patterns for expansion by slippage are recovered for longer times. At 1.0 μm dNTP, the same patterns of band intensities as in Fig. 3 are observed for the first 10 min, but some decreases in long expansions (notably I14and I12) are evident at longer times. At 10 μm, no significant decreases are evident for at least 60 min. Thus, to obtain the same degree of expansion in the presence of proofreading as in its absence, apparently 10 to 100 times as much dNTP is required.
Figure 7Band patterns obtained by extension with proofreading-proficient DNA polymerase KF (exo+) at three different dNTP concentrations. Under incubation conditions like those used for proofreading-deficient KF exo− (Fig. 3), at low (0.2 μm) dNTP concentration, the self-priming sequence 32P(CTG)16(CAG)4 is extended to blunt-ended states in less than 1 min as before, but expansion by slippage is markedly reduced because of strand degradation by proofreading 3′-exonuclease activity. To counteract the degradation, much higher dNTP concentrations are needed. A 5-fold increase (1.0 μm) restores the pattern of expansion by slippage seen in Fig. 3 for the first 10 min; another 10-fold increase (10 μm) is needed to maintain the pattern for the full 60 min.
Single DNA strands containing tandemly repeated CTG or CAG triplets form hairpin structures which, although not as stable as normal DNA duplexes, are nonetheless remarkably stable (
). To investigate the influence that such secondary structures have on strand slippage in polymerase-catalyzed repeat expansions, we have constructed a strand sequence, (CTG)16(CAG)4, forming a series of self-primed hairpins (Fig. 1) to which repeats of CAG triplets can be added by DNA polymerases. This sequence provides an opportunity to measure the influence of hairpin CTG/CTG interactions on primer/template slippage in repeating CAG/CTG regions. By using a DNA polymerase devoid of exonuclease activity (KF exo−), we find that initial hairpin loops (formed at equilibrium) are fully extended to blunt-ended forms in a matter of seconds, so that the rates at which blunt-end loops rearrange by slippage and add more repeats can be measured over a period of minutes to about 1 h (Fig. 3). With this construct, we observe triplet repeat expansions in a series of discrete slippage steps for which rate constants can be evaluated.
Of the various hairpins formed initially (Fig. 1), those with an even number of bases in the bend appear most stable since they are found in the largest amounts (Table I). The series of even-numbered loops fromA0 to A10 account for over 90% of all folded structures. As expected, the most stable loop is the blunt-ended structure A0, whose initial amount is 61% of total structures at 37 °C. The next most stable loops are initially present in amounts of 5–7%, these being the five even-numbered loops, A2 toA10. Surprisingly, each of the latter is as abundant or more abundant than all the odd-numbered loops collectively. The abundance of even-numbered loops does not decrease fromA2 to A10 as might be expected but instead shows a minimum at A4(5%). Possibly the longer overhanging templates, shown simply as (GTC)n* in Fig. 1, form a second hairpin fold providing some additional stability.
The initial amounts of loops formed at equilibrium, in the absence of polymerase, can be used to calculate relative loop stabilities in terms of standard free energy, ΔG o = −RTlnKeq. From the ratio of equilibrium amounts, Keq = 7/61, we findA2 is less stable than A0by RTln(61/7) = 1.3 kcal/mol at T = 37 °C or 310 K. Similarly, from Keq = 2/61,A1 is found to be 2.1 kcal/mol less stable thanA0 and thus 0.8 kcal/mol less stable thanA2. Finding A1 about 1 kcal/mol less stable than A2 is consistent with our observation (Table I, footnote) that slippage by 1 triplet, converting A0 to A1, is an order of magnitude slower than the rate for 2-triplet slippage converting A0 to A2.
As shown in Fig. 6 B, the rate of slippage by 2 triplets may be simply related to the ratio of CTG/CTG interactions to CAG/CTG interactions involved in slippage. The logarithm of the slippage rate constant, when plotted against the ratio (CTG/CTG)/(CAG/CTG) in the blunt-ended hairpin undergoing slippage, shows a linear relationship with a positive slope. The slope indicates that slippage rate increases exponentially with the ratio. The implication is that hairpin CTG/CTG interactions promote slippage by 2 triplets and thereby provide a favorable way of propagating waves of expansion in CAG/CTG repeat regions.
In the expansion of the blunt-ended loops in our system, there are several reasons to think that slippage begins internally with CTG/CTG interactions rather than at the primer 3′ terminus with CAG/CTG interactions. We observe (Fig. 2) that the CTG/CTG region has a melting temperature 24 °C lower than the CAG/CTG region, so that local melting needed to initiate slippage is much more likely to occur in the CTG/CTG region. To initiate slippage by 2 triplets, three CAG/CTG interactions need to be disrupted at the primer 3′-end, but only one next to the CTG/CTG region where two weaker CTG/CTG interactions are more easily disrupted. Slippage by 2 triplets is favored over slippage by 1 triplet, in accordance with even-numbered loops of CTGs being more stable than odd-numbered (Table I). The rate constant for 2-triplet slippage increases with CTG/CTG interactions but decreases with CAG/CTG interactions (Fig. 6 A), so that the rate constant increases exponentially with the ratio of CTG/CTG to CAG/CTG interactions, as seen by the linear plot of log(rate constant) versus this ratio (Fig. 6 B). Furthermore, in the presence of 3′-exonucleolytic proofreading (Fig. 7), as long as there are CTG/CTG interactions present (n = 0–8) and slippage rates are 0.28 min−1 or higher (Table I), less than 1.0 μm dNTP concentrations are needed to restore normal slippage patterns, whereas 10 μm concentrations are needed when CTG/CTG interactions are absent (n = 12 and 14) and slippage rates fall to 0.08 min−1 or lower (Table I).
Since our system is single-stranded and relatively short, it is not certain how many of the points above may apply to long double-stranded DNA molecules in vivo. Nevertheless, with these points in mind, we can propose a simple model (Fig. 8) indicating how triplet repeat expansion may occur when there is local strand separation in a region of triplet repeats. Such separation could arise, for example, by negative supercoiling (
Gellibolian R.
Bacolla A.
) of the kind needed to activate transcription or replication. Once separated in a region of triplet repeats, the strands may fold on themselves to form hairpin loops that are initially opposite each other but because of repetition are able to migrate apart by strand slippage and remain separated for longer periods. While apart, if a nick (single-strand cleavage) occurs opposite one of the loops, that loop can stretch and become linear so that a DNA polymerase, perhaps a proofreading-deficient polymerase involved in DNA repair, may fill in the gap to create an expansion on one strand. After the gap is filled and sealed by ligase, a nick opposite the other loop can lead to expansion on the other strand. According to this model, single-strand loops that become separated and nicked in the presence of polymerase may behave like the simple hairpin system we have studied.
Figure 8Proposed model of strand slippage and repeat expansion in vivo suggested by results of current experiments. The repeating arrows symbolize sequence repeats such as triplets. After strand separation and possible loop migration, a nick (cleavage by endonuclease) on one strand allows the loop on the opposite side to be stretched so that the nicked strand can be extended by DNA polymerase (possibly proofreading-deficient polymerase β in eukaryotes) and sealed by DNA ligase.
Our simple system enables one to evaluate the contributions that individual single-strand interactions make to strand slippage in duplex repeat regions. By using the sequence (CTG)16(CAG)4, we have made an evaluation of CTG/CTG interactions contributing to slippage and expansion of CAG/CTG repeats. Similarly, by using the sequence (CAG)16(CTG)4, a corresponding evaluation can be made for CAG/CAG interactions. Since CAG/CAG interactions are known to be weaker, forming less stable hairpin folds (
), it is of interest to see how much difference this makes in the slippage rate constants. Previously it has been generally considered (
) that the greater the stability of single-strand folding, the greater the probability of strand slippage and expansion in repeat regions. However, it is also possible that a difference in folding stability on opposite strands may be important in determining strand slippage and expansion rates. This may be a reason why repeats of triplets such as GAA/TTC, having only purines on one side and pyrimidines on the other, also expand to give diseases such as Friedrich's ataxia (
Cossee M.
Schmitt M.
Campuzano V.
Reutenauer L.
Moutou C.
Mandel J.-L.
Koenig M.
Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7452-7457
Received in revised form: December 17, 1997
Received: September 29, 1997
DOI: https://doi.org/10.1074/jbc.273.9.5204
© 1998 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
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The gild takes on an antique patina when distressed. It can be used for some some soft edged designs or cross-hatching or just simply cutting back on the visual strength of a solid reflective area.
Several methods all work. Steel wool works well, especially if you want a linear scratch, as it tends to look scratched with fine lines.
A more general non-scratched finish is to lightly rub water-gilded leaf with a damp sponge in the same way you normally use to remove excess leaf from around a design. If you stop halfway, leaving some gold, the result is a pleasant antique finish. Red or brown makes a great backing color for 23ct gold (shown on left).
Distressing and scratching of oil-gilded leaf tends to really kill off the lustre. Instead of a velvety matt finish, it can look dull, however every finish can be useful somewhere. It can also tear pieces of oil-size off the glass leaving small holes . On the lower left is oil-gilded 18ct green gold.
On the right is water-gilded aluminium, rubbed with whiting on a damp cloth and backed with black.


The sweeping indictments allege that a corrupt criminal organization was run out of the office of governor, with those in charge attempting to defraud the people of Connecticut "of the intangible right to the honest services" of state government.
Rowland was not included in the indictments, and was not expected to be included. Prosecutors appear to be pursuing a strategy of a series of indictments over the next weeks and months.
Deputy U.S. Attorney John Durham said the "investigation is very active and ongoing."
Indicted Thursday were Peter Ellef, Rowland's co-chief of staff; his son, Peter Ellef II; and contractor William Tomasso. Also listed in the indictment were two Tomasso companies, Tomasso Brothers and Tunxis Management, as well as the younger Ellef's landscape business, LF Design.
The three men are charged with racketeering, conspiracy, bribery, extortion, money laundering and tax violations. Ellef, Ellef II and Tomasso deny the charges.
Rowland, who resigned July 1 to avoid impeachment and removal from office by the General Assembly, has admitted taking "gifts" from Tomasso, Ellef and others. But he has denied giving anything in return.
"At all times relevant to this indictment, under Connecticut law, the supreme executive power of the state was vested in the governor who, through the Office of the Governor, had the authority and responsibility to appoint department commissioners and key administrators; approve and veto legislation; set the agenda for the Bond Commission; authorize expenditures for certain emergency state work; and otherwise set priorities and direction for the state of Connecticut."
Prosecutors, of course, will have to prove their case against Ellef, Ellef II and Tomasso in court. A trial would not be expected until sometime next year.
But the indictment is an outline of the story prosecutors will tell in court.
It is a story of state contracts awarded to Tomasso, who blessed the elder Ellef with more than $1 million in cash, gold coins, trips, meals and vacations.
In return, it is charged, the elder Ellef steered tens of millions of dollars in state contracts to Tomasso and his companies.
It is a story of a money-laundering operation run out of the younger Ellef's business, with Tomasso funneling money to the elder Ellef through the business.
This is a painful time for Connecticut, an embarrassing time. But no matter how painful and embarrassing, Connecticut needs to know exactly what went wrong before it can move forward.