APOBEC3A deaminates transiently exposed single-strand DNA during LINE-1 retrotransposition
Long INterspersed Element-1 ( LINE-1 or L1 ) derived sequences comprise roughly 17% of human genomic DNA ( Lander et al . , 2001 ) . The vast majority of L1 sequences are 5′ truncated , internally rearranged , or are inactivated by mutations and are unable to mobilize ( i . e . , retrotranspose ) to new genomic locations ( Grimaldi et al . , 1984; Lander et al . , 2001 ) . However , it is estimated that the average human genome contains 80–100 retrotransposition-competent ( i . e . , active ) L1s ( Sassaman et al . , 1997; Brouha et al . , 2003 ) . Active human L1s are approximately 6 kb in length and contain a 5′ untranslated region ( UTR ) , two open reading frames ( ORF1 and ORF2 ) , and a 3′ UTR that terminates in a poly ( A ) tail ( Scott et al . , 1987; Dombroski et al . , 1991 ) . ORF1 encodes an ∼40 kDa protein ( ORF1p ) that binds L1 RNA as a trimer ( Holmes et al . , 1992; Hohjoh and Singer , 1996; Martin et al . , 2003; Khazina and Weichenrieder , 2009 ) . ORF1p also has nucleic acid chaperone activity ( Martin and Bushman , 2001; Khazina and Weichenrieder , 2009 ) . ORF2 encodes an ∼150 kDa protein ( ORF2p ) ( Ergun et al . , 2004; Doucet et al . , 2010; Taylor et al . , 2013b ) with DNA endonuclease ( Feng et al . , 1996 ) and reverse transcriptase ( Mathias et al . , 1991 ) activities . The development of a cultured cell assay definitively demonstrated that both ORF1p and ORF2p are required for efficient L1 retrotransposition ( Moran et al . , 1996 ) . Subsequent biochemical studies revealed that L1 retrotransposition occurs by a mechanism termed target-site primed reverse transcription ( TPRT ) ( Luan et al . , 1993; Feng et al . , 1996; Cost et al . , 2002; Kulpa and Moran , 2006 ) . Ongoing L1 retrotransposition events contribute to intra- and inter-individual genetic variation and , on occasion , can disrupt gene function , leading to sporadic genetic diseases ( Beck et al . , 2011; Hancks and Kazazian , 2012 ) . Given the mutagenic potential of active L1s , it is not surprising that host cells have evolved multiple mechanisms to restrict L1 retrotransposition ( Levin and Moran , 2011 ) . Previous studies revealed that over-expression of the wild-type ( WT ) APOBEC3A cytidine deaminase protein ( A3A ) , but not deaminase-defective A3A mutants , could potently inhibit L1 retrotransposition in cultured human cells ( Chen et al . , 2006; Muckenfuss et al . , 2006; Bogerd et al . , 2006b; Kinomoto et al . , 2007; Niewiadomska et al . , 2007 ) . However , deamination events were not detected in L1 retrotransposition events generated in the presence of WT A3A ( Chen et al . , 2006; Muckenfuss et al . , 2006; Bogerd et al . , 2006b; Kinomoto et al . , 2007 ) . Thus , the molecular mechanism by which WT A3A inhibits L1 retrotransposition has remained a mystery . Here , we employ an in vitro assay ( L1 Element Amplification Protocol or LEAP ) that recapitulates the first strand cDNA synthesis step of the L1 integration to elucidate a mechanism for A3A-mediated inhibition of L1 retrotransposition . In LEAP reactions that include RNase H , an enzyme that specifically degrades the RNA strand of an RNA/DNA heteroduplex , we report that single-strand L1 cDNAs are rendered susceptible to A3A-mediated deamination . These data suggest that newly synthesized L1 cDNAs remain annealed to their mRNA templates after reverse transcription , protecting the L1 cDNA from deamination by A3A . We further demonstrate that engineered L1 retrotransposition events derived from cultured human cells that co-express A3A and the uracil DNA glycosylase inhibitor protein ( UGI ) contain strand-specific , A3A-mediated C-to-U deamination events . Together , these data indicate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration . Moreover , our results provide direct evidence that A3A can deaminate single-strand genomic DNA in human cells .
We used a cultured cell assay ( Moran et al . , 1996; Wei et al . , 2000 ) to determine the extent to which A3A inhibits retrotransposition of LINE elements that differ significantly in their nucleotide sequence and structure ( Figure 1A ) . The retrotransposition indicator cassette mneoI ( Freeman et al . , 1994; Moran et al . , 1996 ) was used to tag the 3′ untranslated regions ( UTRs ) of a human L1 ( pJM101/L1 . 3 ) ( Sassaman et al . , 1997 ) , a natural mouse L1 element ( TGF21 ) ( Goodier et al . , 2001 ) , a synthetic mouse L1 element ( L1SM ) ( Han and Boeke , 2004 ) , and a LINE-2 element from zebrafish ( ZfL2-2 ) ( Sugano et al . , 2006; Figure 1A ) . The mneoI cassette consists of an antisense neomycin phosphotransferase gene ( NEO ) disrupted by an intron , which is in the same transcriptional orientation as the LINE element ( Figure 1A ) . This arrangement ensures that G418-resistant foci arise only if the tagged LINE element undergoes successful retrotransposition ( Freeman et al . , 1994; Moran et al . , 1996 ) . HeLa cells do not express endogenous A3A ( Wissing et al . , 2011 ) , and therefore provide a system to measure directly the effects of ectopically expressed A3A on L1 retrotransposition . 10 . 7554/eLife . 02008 . 003Figure 1 . A3A suppresses retrotransposition of distinct LINEs , but does not specifically inhibit L1 endonuclease activity . ( A ) LINE schematics: L1 . 3 , TGF21 , and L1SM encode two ORFs ( ORF1 and ORF2 , outlined in black and red , respectively ) . ZfL2-2 contains one ORF ( outlined in red ) . Indicated are the endonuclease ( EN ) and reverse transcriptase ( RT ) domains in the respective ORFs . All elements are tagged in their 3’UTRs with the mneoI ( NEO ) indicator cassette . The percent nucleotide identity of TGF21 , L1SM , and ZfL2-2 to human L1 . 3 is indicated below each schematic . Black arrowheads indicate repeated monomeric sequences in the 5′ UTRs of TGF21 , L1SM , and ZfL2-2 . ( B ) A3A inhibits retrotransposition of distinct LINEs: shown is the effect of wild-type A3A ( white bars ) , deaminase-deficient A3A_C106S ( gray bars ) , and β-arrestin ( β-arr ) control ( black bars ) on LINE retrotransposition . The x-axis indicates the LINE element . The y-axis indicates percent retrotransposition; in each case the β-arr control is set to 100% . Data were normalized using a circular NEO expression cassette as detailed in Figure 1—figure supplement 1A . Data are expressed as the mean percent retrotransposition derived from three independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all six technical replicates . ( C ) Retrotransposition assays in 4364a ( wild-type CHO cells ( left ) ) and XR-1 ( XRCC4-deficient CHO cells ( right ) ) : the x-axis indicates experiments conducted with the A3A or β-arr expression vector . The y-axis indicates percent retrotransposition; for each reaction the β-arr control is set to 100% . Gray bars indicate retrotransposition of wild-type L1 ( pAD2TE1 ) and black bars indicate the L1 EN mutant ( pAD136 ) . Data were normalized using the circular NEO control ( Figure 1—figure supplement 1A ) . Data are shown as the mean percent retrotransposition derived from three independent experiments consisting of two or three technical replicates each , with error bars representing the standard deviation among all eight technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 00310 . 7554/eLife . 02008 . 004Figure 1—figure supplement 1 . Additional Control Experiments . ( A ) Strategy for A3A toxicity controls: L1 retrotransposition assays ( top schematic: an engineered L1 plasmid co-transfected with either an A3A or β-arrestin expression vector ) were conducted in parallel with toxicity control assays ( bottom schematic: a linear or circular NEO expression vector co-transfected with either an A3A or β-arrestin expression vector ) . The percentages of G418-resistant colonies relative to the β-arrestin control were determined in both experiments . The results from the toxicity control experiments were used to normalize data from the corresponding L1 retrotransposition assays . ( B ) A3A expression inhibits L1 retrotransposition despite exhibiting some cytotoxicity: HeLa cells were transfected with 1 μg of pJM101/L1 . 3 or pU6i NEO expression vector , and increasing amounts of A3A expression vector ( from 0 . 0 μg to 3 . 0 μg ) . The remaining plasmid mass included the β-arrestin expression vector . Thus , each co-transfection consisted of a total plasmid mass of 4 μg . The x-axis indicates the amount of A3A plasmid present in the co-transfection . The y-axis indicates percent of G418-resistant colonies . Shown are experiments conducted with a circular NEO expression vector ( blue line ) , a linearized NEO plasmid ( red line ) , or pJM101/L1 . 3 ( green line ) . Data are expressed as the mean percent G418-resistant colonies derived from two independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all four technical replicates . ( C ) Normalization controls: the colony counts generated in the retrotransposition assay ( Figure 1—figure supplement 1B ) were normalized using the colony counts generated in the linear or circular NEO control toxicity experiments . The x-axis indicates the amount of A3A plasmid in the co-transfection . The y-axis indicates normalized retrotransposition values using the data with the circular NEO expression vector ( blue bars ) or the linearized NEO plasmid ( red bars ) . Data are expressed as the mean percent retrotransposition derived from two independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all four technical replicates . ( D ) A3A inhibits the retrotransposition of human LINE-1 elements expressed from various contexts: HeLa cells were co-transfected with 0 . 5 μg of an A3A expression vector and 0 . 5 μg of the indicated L1 expression vector per well of a six-well tissue culture plate . The x-axis indicates the L1 expression vector . In pDK101 , a CMV promoter augments L1 . 3 expression from the pCEP4 backbone . In pKS101/L1 . 3 . sv+ , the native L1 5′UTR drives transcription from a pBluescript ( Stratagene ) backbone . In pJM102/L1 . 3 , the CMV promoter drives L1 expression from the pCEP4 episomal backbone; the construct lacks the L1 . 3 5′UTR . The y-axis indicates the normalized percent of retrotransposition in the presence of a β-arrestin ( black bars ) or A3A ( gray bars ) expression plasmid . Data were normalized relative to the linear NEO control . Data are expressed as the mean percent retrotransposition derived from two independent experiments consisting of three technical replicates each , with error bars representing the standard deviation among all six technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 004 We co-transfected HeLa cells with retroelement expression constructs and plasmids encoding WT A3A or a deaminase-deficient mutant , A3A-C106S ( Chen et al . , 2006 ) . As a control we included β-arrestin , which does not significantly affect L1 retrotransposition ( Bogerd et al . , 2006b ) . A3A expression inhibited human L1 . 3 retrotransposition to ∼28% of control levels ( Figure 1B ) , in agreement with previous reports ( Chen et al . , 2006; Muckenfuss et al . , 2006; Bogerd et al . , 2006b ) . In comparison to control levels , A3A also inhibited retrotransposition of TGF21 ( to ∼20% ) , ZfL2-2 ( to ∼17% ) , and L1SM ( to ∼61% ) ( Figure 1B ) . The milder inhibition of A3A on L1SM retrotransposition may be due to the GC-rich nature of the L1SM transcript , the elevated steady state levels of L1SM mRNA , and/or an increased number of L1SM retrotransposition events per cell ( Han and Boeke , 2004 ) . Additional controls revealed that the reduction of G418-resistant HeLa cell foci observed in the presence of A3A reflects specific inhibition of L1 retrotransposition , rather than non-specific cytotoxicity ( Figure 1—figure supplement 1A–C; see Methods and Materials ) . A deaminase-deficient mutant , A3A-C106S , did not significantly restrict retroelement mobility ( Figure 1B ) , in agreement with previous studies ( Chen et al . , 2006; Muckenfuss et al . , 2006; Bogerd et al . , 2006b ) . Moreover , A3A inhibited L1 . 3 retrotransposition when it was expressed from its native 5′UTR on a non-episomal vector ( Figure 1—figure supplement 1D: pKS101/L1 . 3/sv+ ) . Together , these experiments show that A3A inhibits retrotransposition of diverse LINE elements in cultured cells . Since LINE element retrotransposition proceeds by target-site primed reverse transcription ( TPRT ) , which requires element-encoded endonuclease ( EN ) and reverse transcriptase ( RT ) activities ( Luan et al . , 1993; Feng et al . , 1996 ) , we investigated whether A3A specifically interferes with L1 EN or L1 RT activity . In addition to canonical TPRT , human L1s can mobilize by an alternative , endonuclease-independent retrotransposition mechanism ( ENi ) in cells that lack p53 activity and are defective for components of the non-homologous end joining ( NHEJ ) DNA repair machinery ( Morrish et al . , 2002 , 2007; Coufal et al . , 2011 ) . We reasoned that if A3A specifically inhibits L1 EN activity , ENi L1 retrotransposition events should escape A3A inhibition . To test this hypothesis , we co-transfected a human L1 ( either WT pAD2TE1 or EN-deficient pAD136 [Doucet et al . , 2010] ) and a WT A3A expression construct into Chinese Hamster Ovary ( CHO ) cell lines that are NHEJ-proficient ( 4364a ) or NHEJ-deficient ( XR-1 ) ( Morrish et al . , 2002 , 2007 ) . A3A expression inhibited WT L1 retrotransposition in both cell lines , and also inhibited ENi retrotransposition in XR-1 cells ( Figure 1C ) . These data demonstrate A3A can inhibit L1 retrotransposition in a hamster cell line and that the A3A-mediated reduction of L1 retrotransposition is not due to inhibition of L1 EN activity . To test whether A3A inhibits L1 RT activity , we performed in vitro LEAP assays ( Kulpa and Moran , 2006 ) with ribonucleoprotein particle ( RNP ) preparations derived from cells transfected with an engineered L1 construct ( Figure 2A ) . We purified recombinant WT A3A ( rA3A ) ( Figure 2—figure supplement 1A , B ) and deaminase-deficient ( rA3A_C106S ) ( data not shown ) proteins from Escherichia coli . As expected rA3A had cytidine deaminase activity on single-strand ( Figure 2B ) , but not on double-stranded DNA ( Figure 2—figure supplement 1C ) , whereas rA3A_C106S lacked appreciable deaminase activity ( Figure 2B ) . Incorporating increasing amounts of rA3A or rA3A_C106S into LEAP or Moloney murine leukemia virus ( MMLV ) RT reactions did not significantly affect the synthesis of L1 or MMLV-derived cDNA products ( Figure 2C , Figure 2—figure supplement 1D ) . Thus , A3A does not specifically block L1 RT activity . 10 . 7554/eLife . 02008 . 005Figure 2 . Recombinant A3A ( rA3A ) deaminates L1 cDNAs in vitro . ( A ) LEAP assay rationale: L1 RNP preparations consisting of the L1 RNA ( gray ) , L1 ORF1p ( white ovals ) , and L1 ORF2p ( blue oval ) are incubated with a 3′ RACE primer consisting of a unique adapter sequence that contains a single cytidine ( red ) followed by an oligo dT sequence ( black lettering ) . After reverse transcription ( blue arrow ) , the resultant L1 cDNAs ( blue line ) are PCR amplified using primers specific to the engineered L1 and the unique adapter sequence ( green arrows ) . ( B ) Recombinant A3A has deaminase activity in vitro: twofold serial dilutions ( 500 ng–15 . 62 ng ) of WT rA3A ( left panel ) or deaminase-deficient rA3A_C106S ( right panel ) were incubated with a fluorescein isothiocyanate ( FITC ) labeled single-strand DNA oligonucleotide containing a single cytidine residue . The products were treated with recombinant uracil DNA glycosylase ( UDG ) and NaOH and then were resolved by gel electrophoresis . A control reaction was included without recombinant protein ( marked Oligo ) . ( C ) Recombinant A3A does not inhibit L1 RT activity: control LEAP reactions with RNP preparations from HeLa cells transfected with WT ( pDK101 ) , RT- ( pDK135 ) , or EN- ( pJJH230A/L1 . 3 ) human L1s ( upper gel ) . HeLa indicates untransfected HeLa cells; no RNP indicates control reactions lacking RNPs . Increasing amounts of rA3A ( ng ) did not significantly affect LEAP activity . Samples containing a deaminase-deficient rA3A_C106S , a heat killed rA3A ( HK ) , or without LEAP products ( H2O ) served as controls . MMLV RT reactions ( lower gel ) confirm the integrity of purified RNA isolated from RNP preps used in the LEAP assay . Notably , the increased size of the EN- RNP RT products is due to a higher molecular weight product generated from pJJH230A/L1 . 3 , which contains an mblastI indicator cassette instead of an mneoI indicator cassette . Size standards ( bp ) are indicated at the left of the gel . ( D ) Sequence characterization of LEAP Products: shown is the ( + ) strand sequence of the LEAP product . Guanosine nucleotides are indicated in red . Black stars and numbers indicate the frequency of G-to-A mutations ( corresponding to C-to-U mutations in the minus ( − ) strand L1 cDNA ) that occurred on ( + ) strand L1 cDNA . Blue circles indicate other nucleotide changes . The blue An indicates the LEAP product poly ( A ) tail . Blue underlining indicates the L1 3′ end PCR primer . Green underlining indicates the LEAP adapter ( 5np1 ) sequence . Top panel: LEAP products generated under the following conditions: no rA3A protein , 100 ng of wild-type rA3A , 100 ng of deaminase-deficient rA3A_C106S . Bottom panel: LEAP products generated under the following conditions in the presence of RNase H: no rA3A protein , 100 ng of wild-type rA3A , 100 ng of deaminase-deficient rA3A_C106S . One hundred products were characterized for each condition . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 00510 . 7554/eLife . 02008 . 006Figure 2—figure supplement 1 . Control Experiments with Recombinant A3A . ( A ) Purification of rA3A from E . coli by Ni affinity: SDS 4–12% polyacrylamide gel electrophoresis and Coomassie blue staining were used to monitor purification of the His-tagged recombinant A3A ( rA3A ) protein . Input bacterial lysate was loaded onto the Ni-Sepharose column ( Lysate ) . Flow through lysate and wash fractions were collected ( Unbound and Wash , respectively ) . Recombinant A3A was eluted from the column with lysis buffer containing 0 . 5M imidazole and consecutive elution fractions were collected ( F1-4 ) . Most of the His-tagged rA3A was eluted in F2 ( arrow ) . Approximate molecular sizes ( kD ) are indicated at the left of the gel . ( B ) Purification of rA3A by gel filtration: recombinant A3A purified by Ni-Affinity was further purified by gel filtration by fast protein liquid chromatography ( FPLC ) on a Superdex 200 column . Arrows indicate the approximate molecular weights ( kD ) of the proteins . The x-axis indicates the elution volumes and fraction numbers . The y-axis indicates the UV absorbance at 280 nm . ( C ) Recombinant A3A does not deaminate double-stranded DNA: single-strand ( lanes 1 and 7 ) or double-stranded DNA ( lanes 2–4 and 8–10 ) substrates were incubated without ( − ) or with ( + ) rA3A ( 250 ng ) , were treated with UDG , and the products were resolved by gel electrophoresis on 15% polyacrylamide TBE-Urea Novex gels ( Invitrogen ) . The relative ratios of the target ( Oligo ) and complementary ( asOligo ) oligonucleotides are indicated at the top of the figure . A non-specific oligonucleotide ( ns ) was also included as a control ( lanes 6 and 12 ) . As an additional control to rule out potential competition of free asOligo for UDG activity , asOligo was added after rA3A incubation with ssDNA Oligo ( lanes 5 and 11 ) . ( D ) Recombinant A3A does not inhibit MMLV-RT activity: from left to right: MMLV RT reactions using purified RNA isolated from pDK101 ( WT ) or pDK135 ( RT- ) RNPs , untransfected HeLa cell RNPs ( HeLa ) , and a no RNA sample . Recombinant WT rA3A ( 100 ng and 300 ng ) , deaminase-deficient rA3A_C106S ( 300 ng ) , and ‘heat-killed’ rA3A ( 300 ng ) were included in MMLV RT reactions . Size standards ( bp ) are indicated at the left of the gel image . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 00610 . 7554/eLife . 02008 . 007Figure 2—figure supplement 2 . A3A Deamination Events in LEAP products . Each chart lists all possible trinucleotide contexts with a ‘G’ as the middle nucleotide ( corresponding to a ‘C’ on the first-strand LEAP cDNA ) . The first column shows the number of times each trinucleotide appears in the LEAP product sequence . Grayed-out rows indicate that the trinucleotide is not present in the LEAP product sequence . The second column lists how many times each trinucleotide is available in 100 LEAP products . The third column quantifies how many times a deamination event was observed within each trinucleotide . The fourth column quantifies the percent of available sites deaminated in 100 LEAP products . Colored rows below each chart indicate the overall frequency of deamination events of all ‘G’ nucleotides . An additional colored cell , below and to the right of each chart , lists the percent of sequences deaminated at the sole ‘C’ nucleotide in the single-strand oligonucleotide adapter sequence . Specific data points mentioned in the text are highlighted in red on each chart . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 00710 . 7554/eLife . 02008 . 008Figure 2—figure supplement 3 . Distribution of deamination events per LEAP product . The x-axis indicates the number of G-to-A changes; the y-axis indicates the number of LEAP products . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 00810 . 7554/eLife . 02008 . 009Figure 2—figure supplement 4 . Summary of LEAP products generated in the presence of rA3A and RNase H . Individual LEAP products ( first column ) were generated from four independent LEAP reactions , indicated by colored shading . For each product , poly-A tail length ( second column ) , the total number of G-to-A changes ( third column ) , and the total number of other nucleotide changes ( fourth column ) are indicated . Columns G29 through G155 represent the 19 nucleotide positions that were deaminated in WT rA3A-containing LEAP reactions . For each product , an ‘X’ indicates the specific location of a deamination event . The Adapter column indicates deamination events within the single-strand LEAP adapter . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 009 To determine whether A3A can deaminate L1 cDNAs in vitro , we sequenced 100 L1 cDNA products from LEAP reactions conducted in the absence of A3A ( no rA3A ) or in the presence of rA3A or deaminase-deficient rA3A_C106S . We did not detect significant numbers of C-to-U deamination events in L1 cDNAs derived from LEAP reactions in the absence of rA3A or the presence of rA3A_C106S ( Figure 2D ) . In contrast , LEAP products generated in the presence of rA3A contained abundant C-to-U editing that was most frequent at the single 5′-TCA-3′ consensus A3A deamination site ( Chen et al . , 2006 ) present in the single-strand oligonucleotide adapter used to prime L1 RT reactions ( Figure 2D , Figure 2—figure supplement 2 , top chart in the middle panel , 91 of 100 ( 91% ) total 5′-TCA-3′ sites within the 100 single-strand oligonucleotide adapters showed evidence of deamination ) . However , far less deamination was detected within L1 cDNA LEAP products at the six internal 5′-TCA-3′ sites ( Figure 2D , Figure 2—figure supplement 2 , top chart in the middle panel , only 25 of 600 ( 4 . 2% ) 5′-TCA-3′ sites within the 100 L1 cDNA products showed evidence of deamination ) . The non-uniform pattern of deamination events observed above suggested that the L1 cDNA may remain annealed to L1 mRNA , generating L1 mRNA/cDNA heteroduplexes after reverse transcription , thereby leaving only the single-strand oligonucleotide LEAP adapter susceptible to deamination . Thus , we tested whether inclusion of RNase H into LEAP reactions renders L1 cDNA susceptible to deamination . In the presence of rA3A and RNase H , 228 of 600 ( 38% ) 5′-TCA-3′ substrates within the L1 cDNAs showed evidence of deamination ( Figure 2D , Figure 2—figure supplement 2 , bottom chart in the middle panel ) . Importantly , sequencing revealed differing poly ( A ) tail lengths and unique deamination patterns in each of the 100 LEAP products , indicating that they were derived from independent L1 cDNAs ( Figure 2—figure supplement 3 , panel e; Figure 2—figure supplement 4 ) . In contrast , in the presence of rA3A_C106S and RNase H , only 6 . 7% of 5′-TCA-3′ substrates within 100 L1 cDNAs demonstrated evidence of deamination ( Figure 2D , Figure 2—figure supplement 2 , bottom chart in the right panel ) . Similarly , in reactions conducted with RNase H alone , 4 . 2% of 5′-TCA-3′ substrates within 100 L1 cDNAs contained signatures of deamination ( Figure 2D , Figure 2—figure supplement 2 , bottom chart in the left panel ) . The slightly increased number of deamination events observed with RNase H alone may reflect endogenous cytidine deaminase activity present in HeLa RNP preparations . APOBEC3B ( A3B ) might be responsible for these events as it is expressed in HeLa cells and can suppress L1 retrotransposition ( Chen et al . , 2006; Bogerd et al . , 2006b; Wissing et al . , 2011 ) . These results suggest that the L1 cDNA , when annealed to the L1 mRNA in an mRNA/cDNA heteroduplex , is protected from mutation by cellular deaminases . Our results demonstrated that rA3A could deaminate single-strand L1 cDNAs in vitro . We therefore hypothesized that A3A may deaminate transiently exposed single-strand DNA intermediates that arise during L1 TPRT in cultured cells . Uridine residues are efficiently removed from DNA through combined actions of uracil DNA glycosylase ( UNG ) and apurinic/apyrimidinic endonuclease ( APE ) ( Krokan et al . , 2002 ) . In principle , these activities could repair and/or degrade deaminated single-strand DNAs that arise during TPRT . Degradation of deaminated retrotransposition intermediates would reduce L1 retrotransposition levels in cultured cell assays and could , in principle , eliminate evidence of A3A-mediated deamination events in the resultant retrotransposed L1 sequences . Such a scenario could explain why previous studies failed to detect A3A-mediated deamination events in engineered L1 retrotransposition events ( Chen et al . , 2006; Muckenfuss et al . , 2006; Bogerd et al . , 2006b; Kinomoto et al . , 2007 ) . To test whether UNG contributed to the inhibition of L1 retrotransposition downstream of A3A activity , we conducted L1 retrotransposition assays in the presence of codon optimized bacteriophage-derived uracil DNA glycosylase inhibitor ( UGI ) that selectively inhibits UNG activity ( Kaiser and Emerman , 2006; Figure 3A ) . Remarkably , UGI expression from a transfected plasmid ( pLGCX_UGI ) alleviated A3A-mediated L1 inhibition by ∼twofold ( Figure 3B: from ∼25% to ∼57% of control levels ) . In contrast , UGI expression had little effect on L1 retrotransposition activity in assays conducted in the presence of A3A deaminase mutant ( A3A-C106S ) or β-arrestin control ( Figure 3B ) . Similarly , UGI expression did not affect the ability of deaminase-deficient A3B mutants ( Bogerd et al . , 2006b ) to inhibit L1 retrotransposition ( Figure 3—figure supplement 1A: see A3B_N-term and A3B_CS data ) . Thus , UGI expression specifically alleviates a deaminase-dependent mechanism of L1 retrotransposition inhibition . 10 . 7554/eLife . 02008 . 010Figure 3 . UGI expression alleviates A3A-mediated inhibition of L1 retrotransposition and allows detection of deaminated L1 retrotransposition events . ( A ) Experimental strategy: cells ( HeLa , upper left , or U2OS , upper right ) were co-transfected with the indicated expression plasmids and assayed for L1 retrotransposition ( bottom left branch ) or insertion analysis ( bottom right branch ) . ( B ) UGI expression alleviates A3A-mediated retrotransposition inhibition: the x-axis indicates the co-expression vector . The y-axis depicts the efficiency of L1 retrotransposition . Shown are results of experiments in the presence ( black bars ) or absence ( gray bars ) of UGI . The results were normalized as in Figure 1—figure supplement 1A . Data are expressed as the mean percent retrotransposition derived from three independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all six technical replicates . ( C ) DNA sequencing results: the left column indicates the engineered L1 sequence . The top row indicates sequence changes observed in recovered L1 insertions; n indicates the number of characterized retrotransposition events . The total retrotransposed sequence observed ( in bp ) includes the L1 sequence and the mneoI/ColE1 cassette . ( D ) An L1 insertion harboring mismatched TSDs: The pre- ( empty ) and post- ( filled ) L1 integration sites are shown . The truncation point ( bp 5483 ) and structure of the engineered retrotransposed L1 are indicated . Deamination of single-strand genomic DNA in the pre-integration site ( red rectangle ) leads to inexact TSDs in the post-integration site ( red lettering/rectangles ) . ( E ) Stable UGI expression alleviates A3A-mediated L1 inhibition: The x-axis indicates the U2OS cell line . The y-axis depicts the efficiency of L1 retrotransposition . Shown are the effects of wild-type A3A ( white bars ) , A3A-C106S ( gray bars ) , and β-arrestin ( β-arr , black bars ) control on L1 retrotransposition . Data were normalized to controls conducted with a circular NEO expression vector ( Figure 3—figure supplement 1 B–C ) . Data are expressed as the mean percent retrotransposition derived from three independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all six technical replicates . ( F ) DNA sequencing results: The left column indicates the engineered L1 sequence . The top row indicates sequence changes observed in recovered L1 insertions; n indicates the numbers of characterized retrotransposition events . The total retrotransposed sequence observed ( in bp ) includes the L1 sequence and the mneoI/ColE1 cassette . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 01010 . 7554/eLife . 02008 . 011Figure 3—figure supplement 1 . Additional Control Experiments . ( A ) UGI does not alleviate L1 retrotransposition inhibition by APOBEC3B cytidine deaminase mutants ( pK_A3B_Nterm and pK_A3B_CS ) : HeLa cells were transfected with 1 μg each of an APOBEC3 expression vector ( WT pK_A3A , WT pK_A3B , deaminase-deficient pK_A3B_Nterm , deaminase-deficient pK_A3B_CS , or pK_ β-arrestin ) , an L1 vector ( pJM101/L1 . 3 ) , and pLGCX/UGI or pLGCX empty vector . The x-axis indicates the APOBEC3 expression construct . Orange bars: vector only control . The y-axis indicates percent retrotransposition , with the β-arrestin control for each reaction set to 100% . Blue bars: UGI expression . Data were normalized to circular NEO control co-transfections . Data are expressed as the mean percent retrotransposition derived from three independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all six technical replicates . Notably , UGI expression does not affect the ability of the deaminase-deficient A3B mutants ( pK_A3B_Nterm and pK_A3B_CS ) to inhibit L1 retrotransposition . ( B ) Effect of A3A expression on L1 retrotransposition and circular NEO controls in control U2OS cells: control U2OS cells were transfected with a total of 1 . 25 μg of DNA , including 1 . 0 μg of pJM101/L1 . 3 plasmid . For the β-arrestin control transfections , 250 ng of β-arrestin plasmid were used in the experiments . A3A transfections consisted of 0 . 0 ng , 0 . 25 ng , 25 ng , 100 ng , or 250 ng of A3A expression vector and the appropriate amount of β-arrestin plasmid to bring total plasmid mass to 1 . 25 μg . Blue bars indicate pJM101/L1 . 3; green bars indicate the pU6i NEO control . The x-axis shows the amount of A3A plasmid used in the experiment . The y-axis shows percent of G418-resistant colonies , with β-arrestin control co-transfection ( 0 ng A3A ) set to 100% . Data are expressed as the mean percent G418-resistant colonies derived from two independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all four technical replicates . ( C ) Effect of A3A expression on L1 retrotransposition and circular NEO controls in U2OS_UGI cells: co-transfection experiments were carried out as described in Extended Data Figure 3B . Blue bars indicate pJM101/L1 . 3; green bars indicate pU6i NEO control . The x-axis shows the amount of A3A plasmid used in the experiment . The y-axis shows percent of G418-resistant colonies , with β-arrestin control co-transfection ( 0 ng A3A ) set to 100% . Data are expressed as the mean percent G418-resistant colonies derived from two independent experiments consisting of two technical replicates each , with error bars representing the standard deviation among all four technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 01110 . 7554/eLife . 02008 . 012Figure 3—figure supplement 2 . Summary of L1 retrotransposition events generated in U2OS_UGI cells in the presence of β-arrestin . The pJM140/L1 . 3/Δ2/k7 retrotransposition indicator cassette is depicted at the top of the Figure . Features of the plasmid are shown to scale: 5′UTR ( green ) , ORF1 ( light blue ) , inter-orf spacer ( light gray ) , ORF2 ( dark blue ) , 3′UTR ( orange ) , NEO cassette ( purple ) with HSV_tk polyA signal ( maroon ) , SV40 promoter/ori ( pink ) , ColE1 bacterial origin of replication ( red ) , and plasmid backbone sequence between the above mentioned features ( dark gray ) . The name of the cell line from which each insertion was recovered is listed at the left side . The length and structural features of characterized insertions are represented graphically at the right side . The truncation point of each insertion , relative to the pJM140/L1 . 3/Δ2/k7 retrotransposition indicator plasmid ( Gilbert et al . , 2005 ) , is depicted in black . For inversion/deletion containing insertions , the break points are depicted in black , with a double line marking the inversion/deletion junction . The inverted sequence is depicted as a left-facing arrow . Some recovered insertions ended at the restriction site used in the recovery procedure; the sites are named in red lettering ( e . g . , see insertion B1-10 ) . Nucleotide changes from the pJM140/L1 . 3/Δ2/k7 sequence are depicted as follows: stars indicate G-to-A changes; circles indicate any other nucleotide change . Blue shapes indicate changes in non-coding regions; green shapes indicate silent mutations; yellow shapes indicate missense mutations; red shapes indicate nonsense mutations . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 01210 . 7554/eLife . 02008 . 013Figure 3—figure supplement 3 . Summary of L1 retrotransposition events generated in U2OS_UGI cells in the presence of A3A_C106S . Insertion features and nucleotide changes are depicted as described in Figure 3—figure supplement 2 . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 01310 . 7554/eLife . 02008 . 014Figure 3—figure supplement 4 . Summary of L1 retrotransposition events generated in U2OS_UGI cells in the presence of A3A . Insertion features and nucleotide changes are depicted as described in Figure 3—figure supplement 2 . The nonsense mutation G-5390-A recovered from cell line A0-43 is highlighted with a dotted red box . 51 copies of a TTAGGG telomeric repeat , highlighted by the green rectangle , were captured at the inversion/deletion junction of A0-12 . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 01410 . 7554/eLife . 02008 . 015Figure 3—figure supplement 5 . The L1 . 3 G-5390-A mutation decreases L1 retrotransposition efficiency . The G-5390-A mutation was generated by site-directed mutagenesis on wild-type pJM101/L1 . 3 . U2OS_UGI cells were transfected with 1 μg of retrotransposition indicator plasmid . ( A ) Representative retrotransposition assays: from left , wells contain pJM101/L1 . 3 ( wild type ) , pDK135 ( RT-deficient mutant ) , pJM101/L1 . 3/G-5390-A , and an untransfected control . ( B ) Quantification of retrotransposition efficiencies: the x-axis shows the elements used in the assay . RT- indicates the RT-deficient mutant negative control pDK135 . The y-axis indicates the percent of retrotransposition relative to the pJM101/L1 . 3 control . Data are shown as the mean percent retrotransposition derived from two independent experiments consisting of three technical replicates each , with error bars representing the standard deviation among all six technical replicates . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 015 We hypothesized that UGI expression should allow the detection of A3A-mediated C-to-U deamination events in retrotransposed L1s . Thus , we co-transfected HeLa cells with A3A , UGI , and pJM140/L1 . 3/Δ2/k7 , an engineered L1 vector that contains both an mneoI retrotransposition cassette and a ColE1 bacterial origin of replication ( Gilbert et al . , 2002 , 2005 ) ( Figure 3A ) . This modified cassette facilitates recovery of engineered L1 retrotransposition events and their associated flanking genomic DNA sequences as autonomously replicating , kanamycin-resistant plasmids in bacteria ( Gilbert et al . , 2002 , 2005 ) . Characterization of 12 independent retrotransposition events revealed four G-to-A mutations on the L1 coding strand , which correspond to C-to-U deamination events on the ( − ) strand L1 cDNA ( Figure 3C ) . Notably , the 5′ target-site duplication ( TSD ) of one insertion contained a C-to-T change within a 5′-TCA-3′ consensus A3A deamination site in flanking genomic DNA . This putative A3A-mediated deamination event led to an inexact TSD flanking a newly retrotransposed L1 ( Figure 3D ) . To gain further evidence for A3A-mediated deamination of L1 retrotransposition events , we employed a stable UGI-expressing U2OS cell line ( Landry et al . , 2011 ) ( Figure 3A: right side ) . This strategy ensured that UNG activity is inhibited before co-transfection of L1 and A3A expression plasmids ( Landry et al . , 2011 ) . In control U2OS cells , transfection of a low amount of A3A plasmid ( 25 ng ) inhibited retrotransposition to ∼28 . 0% of control levels without substantial cytotoxicity ( Figure 3E , Figure 3—figure supplement 1B ) . By comparison , in U2OS_UGI-expressing cells , A3A only slightly inhibited L1 retrotransposition ( Figure 3E: to ∼83% of control levels; Figure 3—figure supplement 1C ) . Thus , consistent with experiments performed in HeLa cells , stable UGI expression relieves A3A-mediated inhibition of L1 retrotransposition in U2OS cells . We next analyzed sequences of engineered L1 retrotransposition events . We used pJM140/L1 . 3/Δ2/k7 to generate a panel of clonal G418-resistant U2OS_UGI cell lines , isolated the resultant L1 retrotransposition events , and examined them for evidence of A3A-mediated deamination . Characterization of 24 control L1 retrotransposition events did not reveal any G-to-A mutations ( Figure 3F , top panel , Figure 3—figure supplement 2 ) . Similarly , characterization of 30 L1 retrotransposition events generated in the presence of A3A-C106S revealed only five G-to-A mutations ( Figure 3F , middle panel , Figure 3—figure supplement 3 ) . In contrast , characterization of 33 L1 retrotransposition events generated in the presence of A3A revealed 39 G-to-A mutations ( Figure 3F , bottom panel , Figure 3—figure supplement 4 ) . The A3A-induced mutations occurred in a strand-specific manner ( i . e . , we only detected one C-to-T mutation on the L1 coding strand ) , and far outnumbered any other nucleotide changes in retrotransposed products . Notably , one mutation ( pJM140/L1 . 3/Δ2/k7: G-5390-A ) introduced a premature termination codon into L1 ORF2 , which would create a 145 amino acid truncation in ORF2p . This mutation reduced L1 retrotransposition efficiency to ∼10% of control levels ( Figure 3—figure supplement 5 ) . Thus , coupled with identification of a deamination-induced mutation in genomic DNA flanking a newly retrotransposed L1 ( Figure 3D ) , these data suggest that the detected A3A-mediated deamination events occur on single-strand DNA during TPRT rather than on L1 mRNA or single-strand episomal plasmid DNA prior to L1 retrotransposition .
We propose a straightforward model for how A3A inhibits L1 retrotransposition ( Figure 4 ) . In the presence of cellular RNase H , the ( − ) strand L1 cDNA becomes vulnerable to A3A deamination ( Figure 4A ) . Normally , these events would be repaired by combined actions of UNG and APE , which we speculate could lead to the integration of a 5′ truncated L1 cDNA that lacks evidence of deamination . However , in the presence of ectopically expressed UGI , A3A-mediated deamination events become unveiled in the retrotransposed L1s ( Figure 3D , F , 4A ) . We further propose that A3A can act on transiently exposed single-strand genomic DNA flanking L1 integration sites ( Figure 4B , C ) . The deamination of 5′-flanking top-strand genomic DNA could account for generation of inexact target-site duplications ( Figure 3D ) . Interestingly , the structure of the proposed L1 integration intermediate resembles R-loop intermediates , formed during immunoglobulin class switching , that are targeted by an A3A related enzyme , activation-induced cytidine deaminase ( AID ) ( Goodman et al . , 2007 ) . The deamination of 3′ flanking bottom-strand genomic DNA , in principle , could also lead to the generation of inexact TSDs ( Figure 4C ) . While we did not observe these events in cultured cells , we did observe deamination events in the single-strand DNA oligonucleotide primer used to simulate 3′ flanking genomic DNA in LEAP assays ( Figure 2D ) . It is intriguing to speculate that , in the absence of UGI , A3A-mediated editing within 3′ flanking bottom-strand genomic DNA could ultimately lead to loss of the L1 cDNA . Indeed , the ERCC1/XPF endonuclease has been implicated in limiting L1 retrotransposition by cleaving the transiently exposed single-strand 3’ flanking bottom-strand genomic DNA during TPRT ( Gasior et al . , 2008 ) . The DNA lesion resulting from cleavage of this region could then be repaired using the top-strand genomic DNA as a template , leaving no detectable evidence of thwarted L1 retrotransposition events . 10 . 7554/eLife . 02008 . 016Figure 4 . A model for A3A-mediated inhibition of L1 retrotransposition . The left side of the Figure shows L1 integration reactions that occur via TPRT . The right side of the Figure shows the predicted structures of the resultant L1 retrotransposition events . Shown are transiently exposed single-strand genomic DNA regions that ultimately give rise to target site duplications ( TSDs: orange lines ) , the L1 RNA ( red line ) , the L1 cDNA ( blue line ) , and the A3A protein ( green oval ) . UGI expression ( red stop sign ) can inhibit UNG activity . ( A ) Deamination of the L1 cDNA: in the presence of cellular RNase H , A3A-mediated deamination of the L1 ( − ) strand cDNA ( blue lettering ) in the presence of UGI leads to C-to-T mutations on the L1 non-coding strand and G-to-A mutations on the L1 ( + ) coding strand . ( B ) Deamination of the 5′ flanking top-strand genomic DNA: in the presence of UGI , deamination of the transiently exposed single-strand 5′ flanking genomic DNA ( top orange line ) during TPRT results in C-to-T changes in the 5′ TSD relative to the 3′ TSD . ( C ) Deamination of the 3′ flanking bottom-strand genomic DNA: in the presence of UGI , deamination of transiently exposed single-strand 3′ flanking genomic DNA during TPRT , in principle , is predicted to result in a G-to-A change in the 3′ TSD relative to the 5′ TSD . DOI: http://dx . doi . org/10 . 7554/eLife . 02008 . 016 The over-expression of A3A does not abolish L1 retrotransposition completely , but reduces it to ∼30% of control levels . Moreover , some L1 retrotransposition events generated in the presence of A3A and UGI lacked evidence of deamination . Why and how some L1 retrotransposition events evade A3A-mediated deamination requires further study . However , it is possible that ectopic A3A expression is too low to efficiently target L1 TPRT intermediates in some cells or that L1 second-strand cDNA synthesis occasionally is completed before A3A can gain access to TPRT intermediates to deaminate single-strand L1 cDNAs . In sum , we conclude that deamination of transiently exposed single-strand DNA arising during L1 TPRT is responsible , at least in part , for A3A-mediated inhibition of L1 retrotransposition . It is noteworthy that the mobility of an evolutionarily related bacterial group II intron requires RNase H1 activity ( Smith et al . , 2005 ) . Similarly , the related Bombyx mori R2 retrotransposon RT can displace RNA from RNA/DNA hybrids during second-strand cDNA synthesis in vitro ( Kurzynska-Kokorniak et al . , 2007 ) . We hypothesize that annealed L1 mRNA protects the first-strand L1 cDNA , and that cellular RNase H renders these L1 cDNAs vulnerable to A3A deamination during TPRT . Mechanistic insights gained from studying deaminase-dependent A3A-mediated L1 inhibition could be applicable to retroelement inhibition by other A3 proteins . A3A expression is restricted to peripheral blood lymphocytes ( Chen et al . , 2006; Refsland et al . , 2010; Stenglein et al . , 2010; Thielen et al . , 2010; Berger et al . , 2011; Koning et al . , 2011 ) , suggesting that it does not play a physiological role in restricting heritable L1 retrotransposition events . By comparison , A3B , which like A3A localizes to the nucleus , is expressed in human embryonic stem cells ( hESCs ) ( Bogerd et al . , 2006b ) and can restrict engineered L1 retrotransposition in hESCs ( Wissing et al . , 2011 ) . Since heritable L1-mediated retrotransposition events can occur in the early human embryo ( de Boer et al . , 2014; van den Hurk et al . , 2007 ) , A3B may restrict heritable L1 retrotransposition events . Consistent with this notion , recent reports suggest that A3B is expressed at higher levels in human induced pluripotent stem cells ( iPSCs ) when compared to non-human primate iPSCs , and that A3B expression may , in part , account for the lower number of recent L1 insertions in the human genome compared to the chimpanzee genome ( Marchetto et al . , 2013 ) . Intriguingly , an A3B deletion allele also is present at a high allele frequency in some human geographic populations ( Kidd et al . , 2007 ) ; thus , it would be interesting to determine whether the loss of A3B correlates with increased frequency of de novo L1 insertions . APOBEC3 activity has been implicated in genome hyper-mutation in numerous cancers ( Roberts et al . , 2013; Burns et al . , 2013a , 2013b ) . It is interesting to speculate that APOBEC3 expression in cancers may restrict potentially deleterious L1 retrotransposition events , yet may exact a mutagenic cost by contributing to genome hyper-mutation ( Narvaiza et al . , 2012 ) . Indeed , our results suggest that in addition to TPRT , A3A may deaminate any transiently exposed single-strand region of genomic DNA ( e . g . , those arising at replication forks or during transcription ) . This hypothesis is consistent with recent reports indicating that resected single-strand DNA , around DNA breaks , is susceptible to hyper-mutation by APOBEC deamination ( Nik-Zainal et al . , 2012; Roberts et al . , 2012; Taylor et al . , 2013a ) .
All plasmids were grown in DH5α ( F– Φ80lacZΔM15 Δ ( lacZYA-argF ) U169 recA1 endA1 hsdR17 ( rK– , mK+ ) phoA supE44 λ–thi-1 gyrA96 relA1 ) competent E . coli ( Invitrogen , Carlsbad , CA ) . Competent cells were prepared in house as described in Inoue et al . ( 1990 ) . Plasmid preparations were generated using the Qiagen Plasmid Midi Kit ( Qiagen , Germany ) according to the manufacturer's protocol . The pK_βarr , pK_A3A , pK_A3B , pK_A3B_Nterm , and pK_A3B_CS expression plasmids were described previously ( Bogerd et al . , 2006a ) . pK_A3B_Nterm expresses only the N-terminal half of the A3B protein and pK_A3B_CS contains full length A3B with an inactivating point mutation in the C-terminal deaminase active site; both constructs lack deaminase activity in a bacterial mutator assay ( Bogerd et al . , 2006b ) . The previously described deaminase-deficient A3A mutant ( A3A-C106S [Chen et al . , 2006] ) was subcloned into the pK expression vector using HindIII and XhoI restriction sites . All human cultured cell lines employed in this study were authenticated by STR comparative analysis , performed by Genetica DNA Laboratories Inc . , Burlington , NC . The Chinese Hamster Ovary cell lines were used and verified in previous studies ( Morrish et al . , 2002 , 2007 ) . HeLa and U2OS cell retrotransposition assays were carried out as previously described ( Moran et al . , 1996; Wei et al . , 2000 ) . Cells were plated in 6-well dishes ( BD Biosciences , San Jose , CA ) , T-75 flasks ( BD Biosciences ) , or 10 cm dishes ( BD Biosciences or Corning , Corning , NY ) . For retrotransposition assays employing human L1 . 3-based constructs ( including pJM101/L1 . 3 , pDK101 , pKS101/L1 . 3/sv+ , and pJM102/L1 . 3 ) , approximately 1 × 105 cells were plated per T-75 flask or 10 cm dish , and approximately 1 × 103 cells were plated per well in a six-well dish . For assays employing pCEP4/L1SM , approximately 2 × 104 cells were plated per T-75 flask . For assays employing TGF21 and Zfl2-2 , approximately 4 × 105 cells were plated per T-75 flask . Cell plating densities were optimized so that positive controls would yield a quantifiable number of G418-resistant foci ( ∼100–500 foci in a six-well dish and ∼500–2500 foci per 10 cm dish or T-75 flask ) . The reduction of G418-resistant foci in the presence of A3A was determined to be reliable and reproducible . Approximately eighteen hours after plating , transfections were carried out using the FuGene 6 transfection reagent ( Roche , Switzerland ) and Opti-MEM ( Life Technologies ) , according to the protocol provided by the manufacturer ( 3 μl FuGene and 97 μl Opti-MEM per one μg of DNA transfected ) . Media were replaced the following day . Approximately 72 hr post-transfection , cells were subjected to selection with 400 μg/ml G418 ( Life Technologies ) . G418 selection was carried out for 12–14 days and the selection media were replaced every other day . Colonies were washed with 1X phosphate buffered saline ( PBS ) ( Gibco , Carlsbad , CA ) , fixed with 2% paraformaldehyde/0 . 4% glutaraldehyde , and stained with 0 . 1% crystal violet solution . CHO cell retrotransposition assays were carried out in 4364a and XR-1 cell lines as previously described ( Morrish et al . , 2002 ) . Approximately 1 × 105 cells were plated per T-75 flask or 2 × 104 cells per well of a six-well dish . 8 hr later , transfections with wild-type ( pAD2TE1 ) and endonuclease mutant ( pAD136 ) retrotransposition indicator plasmids were carried out using the FuGene 6 transfection reagent and Opti-MEM according to manufacturer's protocol ( 3 μl FuGene and 97 μl Opti-MEM per one μg of DNA transfected ) . Media were replaced the following day . Approximately 72 hr post-transfection , cells were subjected to selection with 400 μg/ml G418 . Selection was carried out for 12–14 days and the selection media were replaced every other day . Colonies were washed with 1X PBS , fixed with 2% paraformaldehyde/0 . 4% glutaraldehyde , and stained with 0 . 1% crystal violet solution . For toxicity control transfections , which were carried out in parallel with each retrotransposition assay , HeLa and CHO cells were plated and transfected as described above , substituting pU6i NEO plasmid for the L1 retrotransposition indicator cassette . As in the retrotransposition assays , selection with 400 μg/ml G418 was initiated approximately 72 hr post-transfection and carried out for 12–14 days . The selection media were replaced every other day . Colonies were fixed and stained as described for the retrotransposition assays . The percent of G418 colonies obtained from A3A/pU6i NEO co-transfections relative to control ( β-arrestin/pU6i NEO ) co-transfections was calculated and used to normalize the number of G418-resistant colonies obtained in the corresponding A3A/L1 and control β-arrestin/L1 retrotransposition assays . In this way , any nonspecific toxicity caused by A3A expression is accounted for , and the percent inhibition reported for each assay reflects specific inhibition of L1 retrotransposition by A3A . WT A3A and deaminase-deficient A3A-C106S cDNAs were inserted into pET28 histidine-tag expression plasmids and expressed in E . coli , strain C43 ( F– ompT hsdSB ( rB- mB- ) gal dcm ( DE3 ) ) ( Lucigen , Middleton , WI ) . Recombinant protein was produced by expression at 37°C in LB plus kanamycin for 3 to 5 hr after induction with 1 mM IPTG . Bacterial cultures were pelleted and cell pellets were frozen prior to lysis . Cell pellets were resuspended in lysis buffer ( 50 mM Tris–HCl [pH8] , 200 mM KCl , 10% glycerol , 1 mM DTT , 35 mM imidazole , and Complete protease inhibitor [Roche] ) and lysed using a fluidizer ( Microfluidics , Westwood , MA ) . The soluble ( supernatant ) fraction was separated by centrifugation at 40 , 000×g for 1 hr at 6°C . Recombinant protein from the supernatant was purified by binding to Ni-Sepharose Fast Flow ( GE Healthcare , United Kingdom ) , washed with lysis buffer , and eluted with lysis buffer containing 0 . 5M imidazole . Protein was concentrated using Amicon Ultracell-10K filters ( Millipore , Billerica , MA ) , further purified by gel filtration on a HiLoad 16/60 Superdex 200 prep grade column ( Amersham , United Kingdom ) , and dialyzed in Slide-A-Lyzer 10K MWCO cassettes ( Pierce , Rockford , IL ) in dialysis buffer ( 50 mM Tris–HCl [pH 8] , 200 mM KCl , 10% glycerol , and 1 mM DTT ) . The proteins then were concentrated using Amicon Ultracell-10K filters ( Millipore ) . Protein purification was monitored by SDS-PAGE , Coomassie blue staining , and immunoblotting with antibodies against His-tag ( Sigma , St . Louis , MO ) and APOBEC3A ( Narvaiza et al . , 2009 ) . UV-spectrophotometry and ImageJ software were used to quantify protein levels . To determine rA3A deaminase activity in UDG-dependent deaminase assays , increasing concentrations of rA3A or rA3A_C106S were incubated with a 5′-end fluorescein isothiocyanate ( FITC ) labeled single-strand deoxyoligonucleotide ( 0 . 4 μM ) in a final reaction volume of 30 μl containing: 40 mM Tris–HCl ( pH 8 . 0 ) 10% glycerol , 40 mM KCl , 50 mM NaCl , 5 mM EDTA , and 1 mM DTT . The reactions were incubated at 37°C for 4–8 hr , stopped by heating to 90°C for 5 min , cooled on ice , and then centrifuged at 10 , 000×g for 1 min . 20 μl of the supernatant were then incubated with uracil DNA glycosylase ( UDG , New England Biolabs ) in buffer containing 20 mM Tris–HCl ( pH 8 . 0 ) and 1 mM DTT for 1 hr at 37°C . The resultant products were treated with 150 mM NaOH for 30 min at 37°C , followed by incubation at 95°C for 5 min , and chilled at 4°C for 2 min . The samples were separated using 15% TBE/urea-PAGE . Gels were directly analyzed using a FLA-5100 scanner ( Fuji , Japan ) . The PAGE-purified single-strand DNA oligonucleotide ( Invitrogen ) used for the deaminase assays ( FITC-TCA ) contains a single cytosine in the A3A specific target trinucleotide , 5′-TCA: ( FITC-5′-TATTATTATTATTATTATTCATTTATTTATTTATTTATTT-3′ ) For deaminase assays on double-strand DNA substrates , the target FITC-TCA oligonucleotide was pre-incubated with either a complementary ( asOligo ) or non-complementary ( ns ) oligonucleotide at the ratios indicated in Figure 2—figure supplement 1C . Preparation of L1 RNPs and LEAP assays were carried out generally as previously described in Kulpa and Moran ( 2006 ) .
Long INterspersed Element-1 ( LINE-1 or L1 ) retrotransposition poses a mutagenic threat to human genomes . Human cells have therefore evolved strategies to regulate L1 retrotransposition . The APOBEC3 ( A3 ) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides ( C-to-U ) in single-strand DNA substrates . Among these enzymes , APOBEC3A ( A3A ) is the most potent inhibitor of L1 retrotransposition in cultured cell assays . However , previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination . Thus , the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic . Here , we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration . These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition .
Transposable elements are often referred to as ‘jumping genes’ because they can move between different locations within a genome . These sequences of DNA are found in many organisms and can make up a significant proportion of the genetic material: almost 50% of the DNA in the case of the human genome . Transposable elements are grouped by how they move to new locations in a genome . Some move by a cut-and-paste mechanism—whereby the transposable element DNA is removed from one location and inserted back at a new genomic location . Others , termed retrotransposons , move by a copy-and-paste mechanism: the DNA sequence is transcribed into an RNA intermediate , and then copied back into DNA before being inserted into a new location . Retrotransposons can accumulate to great numbers in genomes: and one retrotransposon , called LINE-1 , is present at an estimated 500 , 000 copies in the human genome . Although most copies of LINE-1 are inactive , the average human genome contains about 80–100 that are predicted to be able to ‘jump’ to new locations . Given that these retrotransposons could insert into , and disrupt , vital genes , it follows that our cells would have evolved ways to limit their movement . An enzyme named APOBEC3A is known to limit the movement of LINE-1 retrotransposons in cells . APOBEC3A can alter the letters , or bases , that make up the genetic code . This enzyme acts on single-strand DNA to change ‘C’ bases to ‘U’ bases , which could explain how APOBEC3A combats LINE-1 . However , no evidence for such mutation of LINE-1 sequences by APOBEC3A had been found to date . Now , Richardson et al . recreate the copying of LINE-1 RNA back into DNA in a test tube—and reveal that APOBEC3A can mutate single-strand LINE-1 DNA . Critically , as long as the RNA intermediate and DNA copy remain together , the LINE-1 DNA is protected . However , when LINE-1 inserts into a new location the temporarily exposed single strand of LINE-1 DNA becomes susceptible to mutation by APOBEC3A . Human cells can detect and destroy ‘U’ bases in DNA—and only by inhibiting this ability were Richardson et al . able to observe APOBEC3A mutations in new LINE-1 copies within the genomes of living cells . Richardson et al . speculate that the activity of APOBEC3 enzymes must strike a balance between limiting the spread of retrotransposons and minimizing the mutation of the cell's own DNA . Future work could address important questions , such as: do APOBEC enzymes affect the ‘jumping’ of LINE-1 retrotransposons in human reproductive cells and the early embryo , where new LINE-1 insertions could be passed on to subsequent generations ? Also , does a loss of APOBEC3 activity lead to new LINE-1 insertions in cancerous cells ? And does this effect how tumors form and/or progress ? Since APOBEC3 enzymes can cause mutations in cancers , they have been proposed as new targets for anti-cancer drugs—therefore , it is crucial to uncover any harmful effects of inhibiting APOBEC3 enzymes that might limit the effectiveness of such treatments .