cellular genomes are constantly damaged 
 by endogenous agents , such 
 as oxygen radicals formed during aerobic respiration .
this oxidative 
 dna damage is proposed to be a major contributor to carcinogenesis 
 and aging .
one of the most common oxidative dna lesions 
 generated within cells is 8-oxo-7,8-dihydro-2-deoxyguanosine 
 ( 8-oxodg ) .
although structural studies indicate that 8-oxodg induces 
 only minor distortions to the helical structure of dna that are localized 
 to the modified base , 8-oxodg is highly mutagenic due to its dual coding 
 potential . during dna replication ,
8-oxodg 
 adopts either an anti or syn conformation 
 within a polymerase active site . while in the syn conformation , a templating 8-oxodg lesion utilizes the hoogsteen 
 edge of the damaged base to preferentially form base pairs with incoming 
 datp . if left unrepaired , these 8-oxodg : da 
 mispairs will lead to g  t transversion mutations , which have 
 been linked to cancer induction .
although 8-oxodg does not completely block dna synthesis catalyzed 
 by replicative dna polymerases , this lesion does induce the pausing of dna polymerases alpha ( pol ) , 
 delta ( pol ) , and epsilon ( pol ) .
such a stalling event 
 may provide an opportunity for a cell to switch to a dna polymerase 
 that is specialized for the bypass of dna lesions , a process known 
 as translesion dna synthesis ( tls ) .
notably , humans encode four 
 y - family enzymes : dna polymerases eta ( hpol ) , iota ( hpol ) , 
 kappa ( hpol ) , and rev1 ( hrev1 ) .
these four low - fidelity dna 
 polymerases are characterized by a lack of proofreading 3 
  5 exonuclease activity , low processivity , and a relatively 
 spacious , solvent - exposed active site .
importantly , the human y - family 
 enzymes demonstrate a large degree of functional divergence due to 
 the fact that during tls , the nucleotide incorporation fidelity and 
 efficiency of each y - family polymerase is lesion specific .
therefore , it is likely that each y - family polymerase 
 has evolved to catalyze tls of a specific set of lesions .
investigation 
 of the lesion bypass efficiencies and fidelities 
 of the y - family polymerases may help illuminate the in vivo roles of these polymerases for the tls of specific lesions . to this 
 end
, earlier reports have examined the 8-oxodg bypass abilities of 
 hpol , hpol , hpol , and hrev1 separately by using various enzymatic assays .
however , 
 these studies have utilized different reaction conditions and dna 
 substrates to individually investigate each enzyme , complicating direct 
 comparisons between the activities of all four human y - family enzymes . 

furthermore , the kinetic methods previously utilized were unable to 
 accurately measure deletion or insertion events induced by tls .
therefore , 
 we sought to systematically compare the 8-oxodg bypass efficiency 
 of each y - family enzyme under the same reaction conditions and to 
 determine the type and frequency of errors induced by tls of 8-oxodg 
 through a high - throughput short oligonucleotide sequencing assay ( ht - sosa ) 
 that was recently developed in our laboratory .
the ht - sosa method is advantageous for a comprehensive 
 analysis of replication errors induced by tls of a damaged site as 
 this method ( 1 ) provides a complete mutagenic profile of dna damage - induced 
 mutations by accounting for all mutagenic events , such as base deletions , 
 insertions , and substitutions ; ( 2 ) supplies sequencing information 
 for incorporation events opposite , upstream , and downstream from the 
 lesion site ; and ( 3 ) allows for analysis of multibase mutations from 
 a single full - length product . here
, we use a combination of primer 
 extension assays and ht - sosa to investigate for the first time the 
 efficiency and accuracy of 8-oxodg bypass catalyzed by the human y - family 
 dna polymerases both alone and in combination .
optikinase was purchased from usb corporation , 
 dntps were purchased from ge healthcare , and [ -p]atp was purchased from perkinelmer . human pol with a c - terminal 
 his6 tag ,
human pol with an n - terminal his6 tag , and human rev1 with an n - terminal his6 tag were 
 expressed in escherichia coli and purified as previously 
 described . human pol with an n - terminal 
 gst tag was expressed in escherichia coli and purified , 
 and the n - terminal gst tag was subsequently removed by proteolytic 
 cleavage with tobacco etch viral protease as previously described .
the synthetic 40-mer 
 dna template containing 
 8-oxodg ( 40-mer-8oxodg ) was purchased from midland certified reagent 
 company ( table 1 ) .
all other synthetic dna 
 oligomers were purchased from integrated dna technologies ( table 1 and table s1 , supporting information ) .
all dna oligomers were gel purified by denaturing polyacrylamide 
 gel electrophoresis ( page , 10% polyacrylamide , 8 m urea ) .
primers 
 were 5-[p]-radiolabeled by incubating each primer 
 with optikinase and [ -p]atp for 3 h at 37 c . 

dna substrates were generated by annealing a 5-[p]-radiolabeled primer with either the control 40-mer template or 
 damaged 40-mer-8oxodg template at a molar ratio of 1.00:1.15 .
annealing 
 solutions were heat denatured by incubation at 75 c for 5 min , 
 followed by slow cooling to 25 c over several hours .
all kinetic and ht - sosa reactions were 
 performed in optimized reaction buffer r ( 50 mm hepes , ph 7.5 at 37 
 c , 50 mm nacl , 5 mm mgcl2 , 0.1 mm edta , 5 mm dtt , 
 10% glycerol , and 0.1 mg / ml bsa ) at 37 c .
a preincubated solution containing 
 either a single polymerase ( 100 nm ) or all four human y - family polymerases 
 ( 25 nm each ) and 100 nm of a 5-[p]-radiolabeled 
 dna substrate ( 17-mer/40-mer or 17-mer/40-mer-8oxodg ) was rapidly 
 mixed with a solution of all four dntps ( 200 m each ) .
the reaction 
 mixtures were quenched at various times by the addition of 0.37 m 
 edta . a rapid chemical quench flow apparatus ( kintek ) was used for 
 kinetic experiments with reaction times ranging from milliseconds 
 to several minutes .
the reaction products were resolved by using denaturing 
 page and quantified by using a typhoon trio ( ge healthcare ) .
analysis of the running start assays was performed as previously 
 described by calculating the relative lesion bypass 
 efficiencies ( 8-oxodg bypass% ) of each polymerase as a function of 
 reaction time . for each time point t , 8-oxodg
bypass% 
 was calculated as the ratio of the bypass events to the encounter 
 events ( eq 1):1where the total 8-oxodg bypass events ( b ) was the 
 sum of the concentrations of all products with 
 sizes greater than or equal to the 22-mer , and the total 8-oxodg  encounter  
 events ( e ) was the summation of the 21-mer product 
 concentration and the total 8-oxodg bypass events ( b ) .
the 8-oxodg bypass% as calculated by eq 1 was then plotted as a function of reaction time . to compare the 
 8-oxodg bypass efficiency of each polymerase , the t50 was defined as the time required for 
 each y - family polymerase to bypass 50% of the total 8-oxodg lesions 
 encountered .
this t50 value was calculated from the 8-oxodg bypass% plots assuming a constant 
 rate of lesion bypass between the time point immediately before and 
 the time point immediately after the bypass of 50% of the lesion sites 
 encountered .
a preincubated 
 solution 
 of 100 nm or 400 nm of hrev1 and 100 nm of a 5-[p]-radiolabeled dna substrate ( 21-mer/40-mer or 21-mer/40-mer-8oxodg ) 
 was rapidly mixed with a solution containing all four dntps ( 200 m 
 each ) . where indicated , hpol ( 300 nm ) was added to the reactions 
 30 s after the addition of the dntp solution .
lesion bypass products 
 were generated by mixing either the radiolabeled 13-mer/40-mer substrate 
 or the 13-mer/40-mer-8oxodg substrate ( 100 nm ) into a solution containing 
 a single enzyme ( 100 nm ) or all four enzymes ( 25 nm each ) .
this solution 
 was incubated briefly and subsequently mixed with a solution of all 
 four dntps ( 200 m each ) . the reactions containing only hpol 
 or only hpol were incubated at 37 c for 1 h , and the 
 reactions containing only hpol or all 4 human y - family polymerases 
 were incubated at 37 c for 4 h. to produce lesion bypass products 
 with the combination of hrev1 and hpol , either the 16-mer/40-mer 
 or 16-mer/40-mer-8oxodg dna substrate ( 100 nm ) was briefly preincubated 
 with hrev1 ( 100 nm ) and subsequently mixed with a solution containing 
 all four dntps ( 200 m each ) .
after 10 or 60 s for the control 
 or damaged dna substrate reactions , respectively , hpol ( 300 
 nm ) was added , and the reactions were incubated for an additional 
 1 h at 37 c .
after generation of the lesion bypass products , 
 the full - length , newly synthesized dna strands were effectively separated 
 from the template strands by using denaturing page , as the 13-mer 
 and 16-mer primers annealed 5 bases upstream of the 3-end 
 of the 40-mer and 40-mer-8oxodg templates ( scheme 1 ) .
next - generation sequencing libraries 
 were generated by first amplifying each purified , single - stranded 
 dna lesion bypass product by using 1 of 10 unique barcoded primers 
 ( table s1 , supporting information ) and 
 a common reverse primer .
the pcr products were then gel purified , 
 and the remaining adapter sequences required for next - generation sequencing 
 were added in a second round of pcr by using illumina pcr primers 
 1 and 2 ( table s1 , supporting information ) .
the completed sequencing libraries were then gel purified and 
 mixed in equal molar ratios .
the sequencing libraries generated from 
 the lesion bypass products were then mixed with an equal molar amount 
 of bacteriophage x sequencing libraries to increase sequence 
 diversity .
finally , the sequencing library solution was sequenced 
 by using a hiseq 2000 dna sequencer ( illumina ) .
to compare the ability of each 
 human y - family dna polymerase to bypass 8-oxodg under identical in vitro conditions , we carried out running start primer 
 extension assays . to this end
, dna substrates were generated by annealing 
 a 5-[p]-radiolabeled 17-mer primer to either 
 the undamaged 40-mer template or the damaged 40-mer-8oxodg template , 
 which contained a site - specifically placed 8-oxodg lesion ( table 1 and figure 1k ) .
these dna 
 substrates were then extended by the action of a y - family dna polymerase . 

the 8-oxodg lesion did not induce any noticeable stalling of hpol 
 when compared to the undamaged control ( figure 1a and b ) .
thus , the efficiency of hpol nucleotide incorporation 
 was not inhibited by the lesion during the bypass or extension step 
 of 8-oxodg tls .
in contrast , hpol paused during incorporations 
 both opposite 8-oxodg and during the following extension step as indicated 
 by the transient accumulation of the 22-mer and 23-mer products ( figure 1c and d ) .
this result is consistent with previous 
 steady - state kinetic studies indicating that the efficiency of hpol 
 nucleotide incorporation is reduced opposite this lesion and that 
 during the extension step of tls , hpol more efficiently extends 
 the 8-oxodg : da mismatch compared to the correct 8-oxodg : dc base pair .
the 8-oxodg lesion significantly blocked nucleotide 
 incorporation by hpol both opposite the lesion and for the 
 extension step as indicated by the accumulation of the 21-mer and 
 22-mer products , respectively ( figure 1e and 
 f ) .
these two consecutive pause sites were also observed with the 
 control substrate but to a lesser extent , as hpol is known 
 to preferentially misincorporate dg opposite template dt and inefficiently 
 extend the mismatch .
this combination of events reduces hpol 
 activity opposite template dts , a phenomenon known as the t stop .
thus , the relative efficiency of nucleotide 
 incorporation catalyzed by hpol opposite 8-oxodg and during 
 the extension step of tls is predicted to be sequence - dependent .
polymerase 
 hrev1 failed to extend past the lesion site within 20 h ( figure 1h ) .
this failure to produce full - length dna products 
 was expected as hrev1 preferentially functions as a dctp transferase 
 by predominantly incorporating dctp opposite dna lesions or undamaged 
 bases .
nevertheless , the running start 
 assay indicated that the 8-oxodg lesion reduced the activity of this 
 polymerase as hrev1 extended the control dna substrate to a 23-mer 
 but was only capable of extending the damaged dna substrate to a 21-mer 
 product ( figure 1 g and h ) .
running start assays 
 for the human y - family dna polymerases on 
 undamaged and damaged dna templates .
a preincubated solution containing 
 100 nm of 5-[p]-radiolabeled ( a , c , e , g , and 
 i ) 17-mer/40-mer or ( b , d , f , h , and j ) 17-mer/40-mer-8oxodg and either 
 100 nm of the indicated dna polymerase or 25 nm of each y - family polymerase 
 was rapidly mixed with a solution containing all 4 dntps ( 200 m 
 each ) .
the reaction mixtures were quenched at the indicated times 
 with 0.37 m edta and resolved by using denaturing page .
the sizes 
 of important products are indicated , and the 22nd position is denoted 
 with an asterisk ( * ) to indicate an incorporation opposite the 8-oxodg 
 lesion site .
the position 
 of the 8-oxodg lesion within the template strand is indicated by a 
  y.  to determine the effect of 8-oxodg bypass in the presence 
 of all 
 four human y - family dna polymerases in vitro , a running 
 start assay was performed with an equal molar concentration of all 
 four human y - family enzymes ( figure 1i and 
 j ) . the time required for all of the human y - family enzymes to bypass 
 the 8-oxodg lesion was similar to that of hpol alone , suggesting 
 that in the absence of any auxiliary factors , hpol dominates 
 the replication of both undamaged and damaged dna .
furthermore , as 
 with hpol alone , no strong pause sites were observed opposite 
 the lesion ( figure 1j ) .
interestingly , the 
 only strong pause site observed in the presence of all four y - family 
 enzymes was an accumulation of the 18-mer product .
the only y - family 
 enzyme that produced this strong 18-mer product was hrev1 ( figure 1 g and h ) , suggesting that the accumulation of the 
 18-mer was due to a single extension by hrev1 , followed by sequestration 
 of this 18-mer product by bound hrev1 . running start assays performed 
 with the combination of hpol , hpol , and hpol 
 lack this 18-mer product ( figure s1 , supporting 
 information ) , indicating that the accumulation of the 18-mer 
 product in the presence of all four enzymes is indeed due to hrev1 . 

we concluded that when the human y - family polymerases were tested 
 in combination , hpol catalyzed the majority of the nucleotide 
 incorporation events . to define the relative lesion bypass efficiency 
 of each y - family 
 polymerase by using established methods ,
the percent of lesions bypassed by each polymerase with respect 
 to the total number of lesions encountered ( 8-oxodg bypass% ) within 
 the autoradiograms shown in figure 1 was plotted 
 as a function of reaction time ( figure s2 , supporting 
 information ) . to compare the relative lesion bypass efficiency 
 of each enzyme , the time required for each polymerase to bypass 50% 
 of the 8-oxodg sites encountered ( t50 ) relative to the replication of 50% of the corresponding 
 dg sites ( t50 )
this qualitative analysis 
 confirmed that the activity of hpol was not reduced by the 
 8-oxodg lesion .
in contrast , the lesion reduced the nucleotide incorporation 
 rate of hpol and hpol by 2.2- and 4.5-fold , respectively . 

the calculated t50 values 
 also indicated that hpol bypassed the 8-oxodg lesion 186-fold 
 and 1160-fold faster than hpol and hpol , respectively . 

consistently , the efficiency of 8-oxodg bypass by all four human y - family 
 enzymes combined was nearly identical to that of hpol alone . 
 taken together , these data indicate that in the absence of auxiliary 
 factors , hpol bypassed 8-oxodg more efficiently than either 
 hpol or hpol. calculated as the 
 time required 
 to bypass 50% of the 8-oxodg sites .
determined by using the 21-mer/40-mer 
 and 21-mer/40-mer-8oxodg substrates . because hrev1 failed to 
 extend the 17-mer primer past the lesion site within 20 h ( figure 1 h ) , we chose to evaluate the ability of hrev1 to 
 bypass the 8-oxodg lesion by using a standing start primer extension 
 assay .
to this end , an undamaged 21-mer/40-mer or damaged 21-mer/40-mer-8oxodg 
 dna substrate ( figure 2a ) was extended by the 
 action of hrev1 such that the first nucleotide incorporation by hrev1 
 was opposite either undamaged dg or the 8-oxodg lesion site ( figure 2b and c ) .
interestingly , the 8-oxodg lesion reduced 
 the rate of nucleotide incorporation catalyzed by hrev1 opposite the 
 lesion and eliminated the ability of hrev1 to extend the lesion bypass 
 product as indicated by the lack of 23-mer product formation in the 
 presence of 8-oxodg ( figure 2c ) .
the plot of 
 dg and 8-oxodg bypass ( figure 2d ) revealed 
 that 15% of the 8-oxodg lesions were not bypassed within 30 s , compared 
 to only 6% of the undamaged dg sites that were not bypassed within 
 the same time .
this result suggests that approximately 9% of the hrev1 
 that encountered 8-oxodg formed nonproductive complexes due to interactions 
 with the lesion .
analyses of the standing - start t50 values indicated that the 8-oxodg 
 lesion reduced the efficiency of hrev1 nucleotide incorporation by 
 6.8-fold ( table 2 ) . thus , of the four human 
 y - family enzymes , hrev1 was most significantly inhibited by the 8-oxodg 
 lesion relative to undamaged dg .
however , due to the relatively fast 
 rate of nucleotide incorporation of this enzyme , hrev1 was the second 
 most efficient enzyme to bypass 8-oxodg overall and only 1.5-fold 
 less efficient than hpol ( table 2 ) . 
 to examine the ability of hrev1-generated lesion bypass products to 
 be extended , the undamaged
21-mer/40-mer or damaged 21-mer/40-mer-8oxodg 
 dna substrate was extended by the action of either hrev1 alone or 
 in combination with hpol , the y - family enzyme purported to 
 be specialized for the extension step of tls . within 30
s , 
 hrev1 traversed the majority of the dg or 8-oxodg sites as demonstrated 
 by the accumulation of the 22-mer product ( figure 2e and f ) . as previously observed in the running start primer 
 extension assays , these 22-mer products were also not extended to 
 full - length products by the action of hrev1 alone . however
, if the 
 control or damaged substrates were reacted with hrev1 for 30 s to 
 allow for the bypass of the lesion or undamaged dg , followed by the 
 addition of hpol to facilitate the extension of the hrev1-generated 
 lesion bypass products , these 22-mer products were extended to full - length 
 within 1 h. thus , although hrev1 is capable of bypassing the 8-oxodg 
 lesion efficiently , a second polymerase is required for the extension 
 of the lesion bypass products .
standing start assays for hrev1 and hpol 
 on undamaged and 
 damaged dna templates .
the position of the 8-oxodg lesion within the template strand is indicated 
 by a  y.  a preincubated solution containing 
 400 nm of hrev1 and 100 nm of 5-[p]-radiolabeled 
 ( b ) 21-mer/40-mer or ( c ) 21-mer/40-mer-8oxodg was rapidly mixed with 
 a solution of all 4 dntps ( 200 m each ) .
the reaction mixtures 
 were quenched at the indicated times with 0.37 m edta and resolved 
 by using denaturing page .
the control dg or damaged 8-oxodg bypass% was plotted as a function 
 of reaction time .
a preincubated solution containing 100 nm of hrev1 
 and 100 nm of 5-[p]-radiolabeled ( e ) 21-mer/40-mer 
 or ( f ) 21-mer/40-mer-8oxodg was rapidly mixed with a solution of all 
 4 dntps ( 200 m each ) . where indicated , hpol ( 300 nm ) 
 was added to the reaction 30 s after the addition of the dntp solution . 

to examine the patterns and frequencies of 
 mutations induced by tls of 8-oxodg catalyzed by the y - family dna 
 polymerases , we used ht - sosa as summarized in scheme 1 . by using this high - throughput method , greater than two million nucleotide sequences generated 
 from lesion bypass products were analyzed to provide a statistically 
 robust data set ( table s2 , supporting information ) .
as a control for errors induced by the high - throughput sequencing 
 approach , we calculated the average error rates within a control flanking 
 region ( positions 14 to 4 relative to the lesion site ) 
 of each sequence , which was initially derived from the 13-mer or 16-mer 
 primers ( table 1 and scheme 1 ) . the average base insertion , deletion , and substitution 
 frequencies within this flanking region were found to be 0.014% , 0.055% , 
 and 0.080% , respectively .
these rates combined for an overall background 
 error rate of 0.15  0.01% at each template position .
this background 
 error rate was at least 51-fold below the error rate of each enzyme 
 opposite the lesion site and more than 10-fold below the background 
 error rate at virtually every other position analyzed within the undamaged 
 or damaged templates .
as an additional control , using ht - sosa we calculated 
 the average base substitution error rates for hpol , hpol , 
 and hpol replicating the undamaged , control template to be 
 1.8  10 , 1.5  10 , and 1.5  10 , respectively .
these values 
 are similar to the nucleotide misincorporation fidelities of hpol 
 ( 2.0  10 to 2.1  10 ) , hpol ( 3.5  10 to 2.9  10 ) , and hpol ( 9.3  10 to 1.1  10 ) as previously determined by 
 presteady - state kinetic assays with undamaged dna substrates , and as previously measured by ht - sosa .
therefore , ht - sosa is a viable technique for 
 the study of the mutagenic profiles induced by tls of 8-oxodg catalyzed 
 by the error - prone y - family polymerases .
we 
 found that hpol correctly incorporated 
 dctp opposite 8-oxodg in 54.5% of the lesion bypass sequences analyzed 
 ( figure 3 ) .
therefore , hpol was 15.7-fold 
 more error prone opposite the lesion than opposite template dg .
opposite 
 8-oxodg , hpol misincorporated datp ( 41.7% ) over misincorporating 
 dttp ( 1.3% ) or dgtp ( 1.2% ) and rarely generated a base insertion ( 0.1% ) 
 or deletion ( 1.3% ) mutation .
interestingly , this result indicates 
 that even though the nucleotide incorporation rate of hpol 
 opposite undamaged dg and 8-oxodg was almost identical ( figure 1a and b , and table 2 ) , hpol 
 was the second most error prone y - family enzyme opposite the lesion 
 site . to compare the hpol error frequency opposite the 8-oxodg 
 lesion to other template positions
, we examined the errors generated 
 by hpol both three template positions upstream and downstream 
 from the lesion site ( figure 4a and b ) .
this 
 analysis demonstrated that the error rates of hpol were nearly 
 unchanged ( within 1.6-fold ) at these template positions in the presence 
 or absence of 8-oxodg ( tables s3 , s4 and s5 , supporting 
 information ) .
therefore , the lesion only influenced the error 
 rate of hpol during incorporations opposite from 8-oxodg .
comparison 
 of the preferred actions of the human y - family polymerases 
 opposite 8-oxodg or dg .
the relative number of nucleotide incorporations , 
 insertion mutations , or deletion mutations produced by each polymerase 
 or polymerase combination opposite template ( a ) dg or ( b ) 8-oxodg 
 are indicated .
histogram of the relative 
 errors generated by the human y - family 
 dna polymerases as a function of template position .
lesion bypass 
 analysis for ( a and b ) hpol , ( c and d ) hpol , ( e and 
 f ) hpol , and ( g and h ) a combination of all 4 y - family polymerases 
 by using either the undamaged 17-mer/40-mer substrate or the damaged 
 17-mer/40-mer-8oxodg substrate .
the relative number of base insertions 
 ( striped bar ) , substitutions ( black bar ) , and deletions ( white bar ) 
 as a percentage of the total dntp incorporations are indicated at 
 each template position .
y.  the indicated 
 template positions are relative to the 8-oxodg lesion site within 
 the 40-mer-8oxodg template . of the four human y - family enzymes ,
hpol 
 was the most error prone opposite 8-oxodg ( figure 3b ) , preferentially misincorporating datp ( 78.0% ) over correct 
 dctp ( 18.5% ) .
overall , we found that hpol was 14-fold more 
 error prone opposite the lesion when compared to undamaged dg .
interestingly , 
 the hpol error rates at positions downstream from the lesion 
 site were nearly unchanged between the damaged or control templates 
 ( figure 4c and d ) , even though the nucleotide 
 incorporation rate of hpol was reduced at these positions within 
 the primer extension assays ( figure 1d ) . in 
 contrast , at positions upstream from the lesion site , the frequency 
 of base insertion mutations increased by an average of 3.6-fold in 
 the presence of 8-oxodg ( tables s3 , s6 , and s7 , supporting information ) .
however , these insertion mutation 
 events were rare , occurring at an average frequency of only 0.16% , 
 indicating that 8-oxodg seldom altered the fidelity of hpol 
 before the lesion entered the polymerase active site .
ht - sosa analysis indicated that hpol 
 correctly incorporated dctp and incorrectly incorporated da opposite 
 8-oxodg in 65.8% and 21.7% of the sequences analyzed , respectively 
 ( figure 3 ) .
thus , even though 8-oxodg reduced 
 the nucleotide incorporation efficiency of hpol to a greater 
 extent than either hpol or hpol ( table 2 ) , hpol was less error - prone than hpol or hpol 
 opposite the lesion .
overall , hpol generated more errors than 
 either hpol or hpol at nearly every template position 
 ( figure 4e and f ) .
the fidelity of hpol 
 has been shown to be lower opposite template pyrimidines than template 
 purines and to violate normal watson 
consistently , we observed the fidelity of hpol to be the 
 lowest opposite template base dt followed by dc , dg , and finally da 
 when replicating the undamaged template ( figure 4e ) and found hpol to preferentially incorporate dg opposite 
 template dt ( figure s3 , supporting information ) . although hpol produced a significant number of frameshift 
 mutations while replicating the undamaged control template , the average 
 hpol base deletion rate at template positions downstream from 
 the 8-oxodg site increased by an average of 3.0-fold ( tables s3 , s8 , 
 and s9 , supporting information ) .
this increase 
 in the average base deletion error rate of hpol downstream 
 from 8-oxodg indicates that the lesion perturbed nucleotide incorporation 
 by hpol after the lesion exited the polymerase active site . to determine the 
 frequencies and types of mutations induced by 8-oxodg bypass catalyzed 
 by hrev1 , the control 16-mer/40-mer substrate or damaged 16-mer/40-mer-8oxodg 
 substrate
were first reacted with hrev1 to allow for hrev1-mediated 
 incorporation opposite undamaged dg or the 8-oxodg lesion , respectively . 

then , because hrev1 is incapable of completely extending similar dna 
 substrates ( figure 2e and f ) , hpol was 
 subsequently added to extend the dna products to full - length for ht - sosa 
 analysis ( figure 5 ) .
of all the y - family polymerases 
 investigated individually or in combination , we found the combination 
 of hrev1 and hpol was the least error prone opposite 8-oxodg 
 by inserting correct dctp ( 92.3% ) over incorrect datp ( 6.9% ) .
importantly , 
 the standing start primer extension assays indicated that approximately 
 15% of hrev1 formed unproductive complexes with the damaged 21-mer/40-mer-8oxodg 
 dna substrate ( figure 2d ) .
therefore , we hypothesize 
 that hpol catalyzed a portion of these observed datp incorporation 
 events after the liberation of the 21-mer/40-mer-8oxodg substrate 
 from unproductively bound hrev1 .
this finding suggests that the error 
 rate of hrev1 opposite 8-oxodg may be even lower than the error frequency 
 observed here .
nevertheless , this result is consistent with the demonstrated 
 activity of hrev1 as a specialized dctp transferase , preferentially 
 inserting dctp opposite damaged or undamaged templating bases .
thus , we conclude that hrev1 is the best suited 
 y - family polymerase to catalyze error - free tls of 8-oxodg .
interestingly , 
 the combination of hrev1 and hpol produced a significant number 
 of deletion mutations at template positions + 1 and + 2 ( tables s10 
 and s11 , supporting information ) .
the majority 
 of these deletion mutations are double base deletion mutations , suggesting 
 that these double base deletion mutations arose after hrev1 incorporated 
 two dc nucleotides into the primer strand , followed by realignment 
 of the primer strand to loop out the + 1 and + 2 template positions , 
 and subsequent extension by hpol. this proposed loop out mechanism 
 is consistent with the previously reported ability of hpol 
 to extend primers that contain limited sequence complementarity at 
 the 3 primer terminus .
histogram of 
 the relative errors generated by the combination of 
 hrev1 and hpol as a function of template position .
sequence 
 analysis of the lesion bypass products generated by the combination 
 of hrev1 and hpol with ( a ) the undamaged 21-mer/40-mer substrate 
 or ( b ) the 21-mer/40-mer-8oxodg substrate .
the relative number of 
 base insertions ( striped bar ) , substitutions ( black bar ) , and deletions 
 ( white bar ) as a percentage of the total dntp incorporations are shown 
 at each template position .
the template bases are denoted , and the 
 8-oxodg lesion is represented as  y. 
the 
 indicated template positions are relative to the 8-oxodg lesion site 
 within the 40-mer-8oxodg template .
all four human y - family enzymes are present within the nucleus during 
 s - phase .
however , the question of how an individual y - family polymerase 
 is recruited to a stalled replication complex in order to bypass a 
 particular lesion in vivo remains unanswered .
we 
 hypothesize that the inherent properties of each polymerase , such 
 as affinity for the lesion site and lesion bypass efficiency , are 
 predictive of the potential for each enzyme to bypass a particular 
 lesion in vivo . in order to establish a direct competition 
 between the human y - family enzymes for 8-oxodg bypass ,
ht - sosa was 
 performed with an equal molar concentration of all four dna polymerases 
 ( figure 4 g and h ) .
this combination of all 
 four enzymes misincorporated datp ( 50.0% ) over correctly incorporating 
 dctp ( 43.5% ) , misincorporating dgtp ( 1.2% ) or dttp ( 1.3% ) , or generating 
 a deletion mutation ( 4.0% ) or an insertion mutation ( 0.6% ) .
therefore , 
 the fidelity of lesion bypass by the combination of all four human 
 enzymes most closely resembled the 8-oxodg bypass fidelity of hpol 
 alone ( tables s4 , s5 , s12 and s13 , supporting 
 information ) .
consistent with the running start primer extension 
 assays ( figure 1i and j ) , we concluded that 
 in the presence of all four human y - family polymerases , hpol 
 catalyzed the majority of nucleotide incorporation events , including 
 opposite the lesion site .
therefore , even though hpol is not 
 the best suited human y - family member to catalyze error - free tls of 
 8-oxodg , the properties hpol allow this polymerase to outcompete 
 hpol , hpol , and hrev1 for bypass of 8-oxodg .
the highly mutagenic 8-oxodg lesion is one of 
 the most prevalent 
 lesions induced by oxidative dna damage . in this article ,
we systematically 
 compared the 8-oxodg bypass efficiencies and fidelities of all four 
 human y - family dna polymerases for the first time in order to predict 
 the enzyme(s ) that have evolved to catalyze tls of 8-oxodg in vivo .
we conclude that ( 1 ) hpol outcompetes hpol , 
 hpol , and hrev1 for nucleotide incorporations opposite 8-oxodg in vitro ; ( 2 ) hpol is the only y - family member to 
 traverse 8-oxodg with the same efficiency as undamaged dg ; ( 3 ) the 
 efficiency of nucleotide incorporation by hrev1 is most significantly 
 reduced opposite the lesion when compared to incorporations opposite 
 undamaged dg ; ( 4 ) hrev1 fails to extend 8-oxodg lesion bypass products ; 
 and ( 5 ) the combination of hrev1 to bypass 8-oxodg and a second polymerase 
 to extend the lesion bypass product is the most accurate polymerase 
 combination to perform tls of 8-oxodg , followed by hpol , hpol , 
 and hpol individually .
the relative 8-oxodg bypass efficiencies 
 based upon the calculated t50 values of the human y - family 
 polymerases are hpol > hrev1  hpol  
 hpol ( table 2 ) .
these findings agree 
 with a previous report investigating 8-oxodg bypass by hpol , 
 hpol , and hpol by using a gapped dna substrate strand 
 displacement assay , although hrev1 was 
 not included in that study .
importantly , we found that the ability 
 of hrev1 to perform the extension step of tls was almost completely 
 eliminated by 8-oxodg ( figure 2c and f ) .
therefore , 
 even though 8-oxodg is efficiently bypassed by hrev1 , a switch to 
 a second polymerase is required for the extension step .
in fact , this 
 inherent inability of hrev1 to extend the lesion bypass products may 
 promote polymerase switching in vivo . if the switching 
 does not occur , the extension of any lesion bypass product by hrev1 
 would result in one or more dctp incorporations into dna due to the 
 function of hrev1 as a dctp transferase , likely leading to the substitution 
 or frameshift mutations .
thus , we hypothesize that the mostly error - free 
 extension of lesion bypass products generated by hrev1 would be carried 
 out in vivo by a second polymerase , such as hpol or dna polymerase  , a b - family polymerase that is also suitable 
 for the extension step of tls . by using ht - sosa , we found that the most common mutation induced 
 by 8-oxodg bypass catalyzed by all four human y - family polymerases 
 either alone or in combination was the incorporation of da opposite 
 8-oxodg ( figure 3b ) .
these results are consistent 
 with previous kinetic studies indicating the predominant substitution 
 catalyzed by hpol , hpol , and hpol opposite 8-oxodg is the misincorporation of datp .
this miscoding 
 potential of 8-oxodg is strongly influenced by the ratio of the anti and syn conformations of 8-oxodg within 
 a polymerase active site .
interestingly , this dual coding potential 
 of lesions such as 8-oxodg is irrelevant to nucleotide incorporations 
 by hrev1 , as structural studies indicate this enzyme uses a novel 
 mechanism whereby incoming dctp interacts and forms two hydrogen bonds 
 with the side chain of arg357 within the n - digit domain of hrev1 , 
 rather than the templating base .
this 
 dctp transferase activity is also supported by kinetic analysis demonstrating 
 hrev1 preferentially incorporates dctp opposite any templating base , 
 damaged or undamaged . here
, we found that the combination 
 of hrev1 and hpol was nearly error - free opposite 8-oxodg ( figure 3b ) .
indeed the low frequency of datp incorporation 
 ( 6.9% ) detected by ht - sosa may be due to the displacement of unproductively 
 bound hrev1 by active hpol , which in turn bypassed the lesion 
 and predominantly incorporated datp opposite 8-oxodg .
therefore , hrev1 
 may be almost completely error free when bypassing 8-oxodg . in this study
, we demonstrate that when all four human y - family 
 polymerases are tested in combination , hpol outcompetes hpol , 
 hpol , and hrev1 for tls of 8-oxodg ( table 2 ) .
notably , previous studies indicate that among the human 
 y - family polymerases , hpol possesses the highest efficiency 
 of nucleotide incorporation into undamaged dna substrates and a 2- to 5-fold greater affinity for undamaged 
 dna substrates when compared to that of hpol , hrev1 , or hpol. furthermore , it has been shown 
 that the presence of 8-oxodg as a templating base does not reduce 
 the binding affinity of either yeast pol or the model y - family polymerase dpo4 for a dna substrate , suggesting that the y - family polymerases 
 are well suited to accommodate this small lesion within their relatively 
 large active sites , without a reduction in dna binding capacity .
thus , 
 the superior dna binding affinity of hpol likely contributes 
 to its dominance within the four enzyme combination assays ( figures 1i and j ) .
however , differences in dna binding affinity 
 alone are not sufficient to explain the 100- to 100,000- fold difference 
 in the t50 values between 
 hpol and the other three y - family dna polymerases ( table 2 ) .
instead , we conclude that a superior nucleotide 
 incorporation efficiency of hpol is the primary contributor 
 to the dominance of hpol for 8-oxodg bypass .
importantly , we 
 chose to test the abilities of the human y - family polymerases to bypass 
 8-oxodg at equal molar concentrations in order to gain insight into 
 the intrinsic abilities of these polymerases to compete for tls of 
 8-oxodg .
however , the relative in vivo expression 
 profiles of the human y - family polymerases under conditions of oxidative 
 dna damage are not known . indeed
the expression of each of the y - family 
 polymerases may not be similar between cell types or may be dynamic 
 during the cell cycle .
further studies are especially needed to elucidate 
 the regulation of the expression and activities of the y - family polymerases 
 in response to cellular dna damage in vivo , which 
 remains an important , unanswered question .
eukaryotes utilize 
 a number of mechanisms to reduce the mutagenic 
 consequences of 8-oxodg , including ( 1 ) excision of the damaged base 
 from 8-oxodgdc pairs by 8-oxoguanine dna glycosylase 1 ( ogg1 ) 
 to initiate base excision repair ( ber ) , ( 2 ) excision of the da base 
 from 8-oxodg : da mispairs by myh adenine glycosylase to facilitate 
 a new incorporation event opposite the lesion , and ( 3 ) hydrolysis 
 of 8-oxodgtp to 8-oxodgmp by mth1 to prevent incorporation of the 
 damaged dntp into replicating dna . despite these important repair 
 mechanisms ,
8-oxodg lesions do persist during s - phase and are encountered 
 by the replication fork .
the majority of mutations induced by 8-oxodg in vivo are g  t transversion mutations , indicating that the error - prone tls of 8-oxodg principally results 
 in datp misincorporation .
our finding that hpol outcompetes 
 hpol , hpol , and hrev1 for nucleotide incorporation opposite 
 8-oxodg is most consistent with a model wherein hpol is the 
 predominant y - family member to catalyze the error - prone bypass of 
 8-oxodg in vivo .
however , in the absence of functional 
 pol , another y - family polymerase may catalyze the bypass 8-oxodg . 

consistently , plasmid - based mutation assays conducted within wild - type 
 or hpol-deficient human cell lines demonstrated that in the 
 absence of hpol , the action of an unknown polymerase increased 
 the total 8-oxodg - induced mutation frequency by 1.2- to 2-fold , primarily due to an increase in g  t transversion mutations . 

our data suggest that hpol is the unknown polymerase since 
 it is the only y - family enzyme which misincorporates datp opposite 
 8-oxodg with a higher frequency than hpol ( figure 3b ) .
the elucidation of which tls - specialized 
 polymerases are responsible 
 for the error - free or error - prone bypass of specific types of dna 
 lesion sites in vivo remains an unanswered and challenging 
 question .
here , we used primer extension assays paired with the recently 
 established ht - sosa approach to directly compare the efficiency and 
 fidelity of 8-oxodg bypass catalyzed by each of the human y - family 
 polymerases individually and in combination .
overall , our data indicate 
 that even though hpol is error - prone opposite 8-oxodg , the 
 superior efficiency of hpol for tls of 8-oxodg suggests that 
 this enzyme is the primary y - family enzyme to catalyze the bypass 
 of 8-oxodg in vivo .
however , we can not completely 
 rule out a role for the other three y - family members for tls of 8-oxodg in vivo . indeed , many dna polymerases may catalyze the bypass 
 of 8-oxodg lesions in vivo to different extents . 

the comprehensive biochemical studies reported here will provide a 
 basis for further evaluation of these possibilities by in 
 vivo studies .
 one 
 of the most common lesions induced by oxidative dna damage 
 is 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg ) . 
 replicative 
 dna polymerases poorly traverse this highly mutagenic lesion , suggesting 
 that the replication fork may switch to a polymerase specialized for 
 translesion dna synthesis ( tls ) to catalyze 8-oxodg bypass in vivo . here , we systematically compared the 8-oxodg bypass 
 efficiencies and fidelities of the tls - specialized , human y - family 
 dna polymerases eta ( hpol ) , iota ( hpol ) , kappa ( hpol ) , 
 and rev1 ( hrev1 ) either alone or in combination . 
 primer extension 
 assays revealed that the times required for hpol , hrev1 , hpol , 
 and hpol to bypass 50% of the 8-oxodg lesions encountered ( t50bypass ) were 0.58 , 0.86 , 108 , and 
 670 s , respectively . 
 although hrev1 bypassed 8-oxodg efficiently , 
 hrev1 failed to catalyze the extension step of tls , and a second polymerase 
 was required to extend the lesion bypass products . a high - throughput 
 short oligonucleotide sequencing assay ( ht - sosa ) 
 was used to quantify 
 the types and frequencies of incorporation errors produced by the 
 human y - family dna polymerases at and near the 8-oxodg site . 
 although 
 hpol bypassed 8-oxodg most efficiently , hpol correctly 
 incorporated dctp opposite 8-oxodg within only 54.5% of the sequences 
 analyzed . in contrast 
 , hpol bypassed the lesion least efficiently 
 but correctly incorporated dctp at a frequency of 65.8% opposite the 
 lesion . 
 the combination of hrev1 and hpol was most accurate 
 opposite 8-oxodg ( 92.3% ) , whereas hpol alone was the least 
 accurate ( 18.5% ) . 
 the t50bypass value and correct dctp incorporation frequency in the presence of 
 an equal molar concentration of all four y - family enzymes were 0.60 
 s and 43.5% , respectively . 
 these values are most similar to those 
 of hpol alone , suggesting that hpol outcompetes the 
 other three y - family polymerases to catalyze 8-oxodg bypass in vitro and possibly in vivo . 
Introduction
Experimental
Procedures
Results
Discussion