translesion dna synthesis ( tls)1 is a significant dna damage tolerance mechanism to overcome replication blocks caused by dna lesions or to seal gaps opposite lesions ( 1,2 ) . in the past decade , numerous dna polymerases ( pols ) have been discovered that possess lesion bypass activity . recently , a new human dna pol has been identified , poln or pol  , and falls in the a - family of dna pols ( 3 ) . this family includes escherichia coli ( e. coli ) pol i and human pol . pol  is a moderately processive enzyme and is able to carry out strand displacement dna synthesis with much higher efficiency than the klenow fragment , the pol - proficient portion of e. coli pol i. it also replicates nondamaged ( nd ) dna with low fidelity ; specifically , it frequently misincorporates dt opposite template dg with about half the efficiency of incorporation of the correct nucleotide dc opposite template dg ( 4,5 ) . because low fidelity of dna synthesis is common for tls pols and pol  lacks 35 exonuclease proofreading activity , a role of pol  in the bypass of specific dna lesions has been proposed ( 4,5 ) . indeed , this pol can carry out tls in vitro , being specifically proficient in accurate bypass of thymine glycols , major groove dna lesions ( 5 ) . however , it is completely blocked by a number of other dna modifications , including a cisplatin - induced gpg intrastrand cross - link , an abasic site , a cyclobutane pyrimidine dimer , and a thyminethymine 6 - 4 photoproduct ( 5 ) . 1abbreviations : tls , translesion dna synthesis ; pol , polymerase ; e. coli , escherichia coli ; nd , nondamaged ; bpde , benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide ; pcl , dnapeptide cross - link ; icl , interstrand dna cross - link ; da , 1,n - etheno - da . abbreviations : tls , translesion dna synthesis ; pol , polymerase ; e. coli , escherichia coli ; nd , nondamaged ; bpde , benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide ; pcl , dnapeptide cross - link ; icl , interstrand dna cross - link ; da , 1,n - etheno - da . a function of pol  in dna cross - link repair in mammalian cells has been proposed ( 6 ) . in particular , the data suggested that pol  is involved in homologous recombination in response to various dna cross - linking agents and potentially interacts with multiple proteins in the fanconi anemia pathway , which are relevant to dna cross - link repair . however , a cellular role for pol  in homologous recombination - independent , tls - dependent repair of dna cross - links has not yet been addressed . additionally , as already mentioned , thymine glycol is the only lesion that pol  has been reported to be able to bypass in vitro ( 5 ) . to understand the cellular function of pol  in tls , it is crucial to identify types of dna lesions that this pol can bypass . because it has been previously established that both the exo and the exo klenow fragment can catalyze tls past certain major groove dna adducts ( 7,8 ) , we hypothesized that in addition to thymine glycols , pol  may catalyze replication bypass of additional major groove dna lesions this study was designed to test this hypothesis to facilitate insight into the cellular function of pol  in tls . p-6 bio - spin columns were obtained from bio - rad ( hercules , ca ) . uracil dna glycosylase , t4 dna ligase , and t4 polynucleotide kinase were purchased from new england biolabs ( beverly , ma ) . slide - a - lyzer dialysis cassettes with a molecular weight cutoff of 10000 were purchased from thermo scientific ( rockford , il ) . yeast pol  and pcna were purified as previously described ( 9 ) and were generous gifts from dr . the lys - trp - lys - lys and lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr peptides were obtained from sigma - genosys ( st . louis , mo ) . ( a ) all nd oligodeoxynucleotides were synthesized by the molecular microbiology and immunology research core facility at oregon health & science university ( portland , or ) . ( b ) oligodeoxynucleotides containing -hydroxypropano - da or -hydroxypropano - dg were synthesized as previously described ( 10 ) . dnas containing dnapeptide cross - links ( pcls ) ( figure 1a , c ) were prepared by the reaction of the -hydroxypropano - da- or -hydroxypropano - dg - containing oligodeoxynucleotides with either 20 nmol of lys - trp - lys - lys or 4 nmol of lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr in the presence of 50 mm sodium cyanoborohydride according to the previously published procedure ( 11 ) . the sequences of oligodeoxynucleotides containing a n - da pcl and a n - dg pcl are shown in table 1 , column 2 , rows 1 and 2 , respectively . structures of dna adducts . the pcls consist of either a tetra ( lys - trp - lys - lys ) or dodecyl ( lys - phe - his - glu - lys - his - his - ser - his - arg - gly - tyr ) peptide attached via an acrolein moiety at the n position of da ( a ) or n position of dg ( c ) . the resulting pcls are referred to as n - da pcl4 , n - da pcl12 , n - dg pcl4 , and n - dg pcl12 . ( f ) structure of ( + ) -trans - anti - bpde - da . a 3-glycerol unit ( gl ) or a dideoxycytidine ( dd ) were incorporated to prevent dna synthesis off of the shorter strand of the icl - containing oligodeoxynucleotides . the number in the parentheses in front of the primer sequence indicates the position of the primer 3-oh . ( c ) the synthesis of the oligodeoxynucleotides containing interstrand dna cross - link ( icl ) between n - das of complementary strands ( figure 1b ) is described in the supporting information . the sequences of a n - da icl1 and a n - da icl2 are shown in table 1 , column 2 , rows 3 and 5 , respectively . the n - da icl2 oligodeoxynucleotide was generated from a precursor oligodeoxynucleotide ( pre - n - da icl2 , table 1 , column 2 , row 4 ) as described previously ( 12 ) . ( d ) oligodeoxynucleotides containing an icl between n - dgs ( figure 1d ) were synthesized as previously described ( 1214 ) , and the sequences of the oligodeoxynucleotides containing each n - dgn - dg icl ( n - dg icl1 , n - dg icl2 , n - dg icl3 , and n - dg icl4 ) are shown in table 1 , column 2 , rows 69 . a detailed description of each type of n - dg icls has been reported previously ( 12 ) . ( e ) an oligodeoxynucleotide containing 1,n - etheno - da ( da ) ( figure 1e and table 1 , column 2 , row 10 ) was synthesized by the molecular microbiology and immunology research core facility at oregon health & science university ( portland , or ) . ( f ) an oligodeoxynucleotide ( 5-ctctcacttcc-3 ) containing ( + ) -trans - anti - benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide - da [ ( + ) -bpde - da ] ( figure 1f ) at the underlined nucleotide was synthesized as previously described ( 15 ) and was a generous gift from dr . this oligodeoxynucleotide was ligated to 16-mer oligodeoxynucleotide ( 5-attccgtatccatttt-3 ) using 800 units of t4 dna ligase at 12 c overnight in the presence of 23-mer scaffold oligodeoxynucleotide ( 5-tggatacggaatggaagtgagag-3 ) . the ligation reaction was terminated by the addition of an equal volume of formamide dye solution [ 95% ( v / v ) formamide , 10 mm edta , 0.03% ( w / v ) xylene cyanol , and 0.03% ( w / v ) bromphenol blue ] , and the products were separated on a 10% acrylamide gel containing 8 m urea . dna was eluted from the gel with 0.5 m ammonium acetate/10 mm magnesium acetate solution , dialyzed against 10 mm tris - hcl ( ph 7.4 ) buffer containing 1 mm edta in slide - a - lyzer dialysis cassette ( mwco of 10000 ) , and further purified through a p-6 bio - spin column . the resulting 27-mer was used for dna replication assays ( table 1 , column 2 , row 11 ) . for replication assays , primers were designed for reactions under a running or standing start condition . in the former case , the primer 3-oh was positioned several nucleotides upstream of the lesion site . in latter case , the primer 3-oh was positioned immediately prior to the lesion site ( 1 primer ) . dna replication assays were carried out in the following buffers : for pol  , the reaction mixture contained 25 mm tris - hcl ( ph 7.5 ) , 8 mm mgcl2 , 10% glycerol , 100 g / ml bovine serum albumin , and 5 mm dithiothreitol . for reaction with pol  with n - dgn - dg icls , mg - acetate was used instead of mgcl2 . for pol  , the reaction mixture contained 40 mm hepes - koh ( ph 6.8 ) , 10% glycerol , 200 g / ml bovine serum albumin , 1 mm dithiothreitol , and 8 mm mgcl2 . dna substrate , pol , and dntp(s ) concentrations , as well as reaction times , are given in figure legends . reaction products were resolved through 15% acrylamide gel containing 8 m urea and visualized using a phosphorimager screen . the percentage of primer extended in extension reactions was determined by taking the ratio of extended primer beyond the adducted base to the total amount of primer ( unextended primer + extended primer up to and opposite the lesion + extended primer beyond the lesion ) . the percentage of primer extended in single nucleotide incorporations was determined by taking the ratio of extended primer to the total amount of primer ( unextended primer + extended primer ) . to address the hypothesis that pol  can catalyze replication bypass of additional major groove dna lesions , we utilized n - da acrolein - mediated pcls as a model for the bulky major groove dna adducts ( 11 ) . specifically , tetra- and dodecylpeptide cross - links ( figure 1a , n - da pcl4 and n - da pcl12 , respectively ) were used . the molecular masses of these pcl - modified bases were 763 da for n - da pcl4 and 1738 da for n - da pcl12 versus 135 da for unmodified adenine . these lesions were positioned 15 nucleotides from the 5 end of the 30-mer template and annealed with a 16-mer radiolabeled primer in which the 3-oh was three nucleotides upstream of the lesion site . data in figure 2a revealed that pol  could fully extend primers annealed to these adducted templates ; under the conditions used , very minimal blockage to dna synthesis was observed one nucleotide prior to the lesion . to assess the identity of the nucleotides incorporated opposite the lesion , single nucleotide incorporations were conducted . because n - da pcl12 is bulkier than n - da pcl4 , further assays used only substrate containing this lesion . data showed that pol  incorporated only the correct nucleotide , dt , opposite n - da pcl12 ( figure 2b ) , suggesting that it catalyzed error - free bypass of this adduct . it is of interest to note that two nucleotides were incorporated for both the nd and the n - da pcl12 substrates when only dttp was present . it has been reported that pol  catalyzes error - prone insertion of dt opposite dg ( 4,5 ) . because the templating nucleotide immediately downstream of the adducted base is a dg , this specific sequence context likely accounts for the observed incorporation of the second dt . to quantitatively evaluate the extent of the blockage posed by n - da pcl12 for the pol -catalyzed nucleotide insertion , dttp titration assays ( 6.4 nm to 100 m dttp at 5-fold incrementally increased concentrations , left to right ) these results demonstrated that pol  could incorporate approximately an equal amount of dt opposite the adducted base at concentrations only 5-fold higher relative to nd da ; specifically , 20% of primers were extended at 800 nm dttp using nd substrate and at 4 m dttp using damaged substrate . replication bypass of n - da peptide cross - links by human pol . ( a ) primer extensions were catalyzed by 50 nm human pol  under running start conditions ( 3 primer ) in the presence of 5 nm primer template containing nd , n - da pcl4 , or n - da pcl12 for 30 min at 37 c . ( b ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol  under standing start conditions ( 1 primer ) in the presence of 2 nm primer template containing nd or n - da pcl12 for 15 min at 37 c . reactions shown in lanes 16 and lanes 712 were conducted side - by - side , and products were separated on the same gel . the reactions shown in panels a and b were carried out in the presence of 100 m individual or all dntps . ( c ) dttp titration assays with 2 nm primer template were catalyzed by 5 nm human pol  under standing start conditions ( 1 primer ) for 10 min at 37 c using the same substrates as described in panel b. dttp concentration ranges from 6.4 nm to 100 m ( 5-fold incremental increase ) . reactions shown in lanes 18 and lanes 916 were conducted side - by - side , and products were separated on the same gel . having demonstrated that pol  can readily bypass n - da pcls , we hypothesized that it may also be able to carry out tls past other n - da major groove adducts , including n - dan - da icls ( figure 1b ) . although icls had been previously thought to pose an insurmountable block to replication , our laboratory has recently demonstrated that human dna pol  and e. coli dna pol iv can catalyze replication bypass of an icl between the n - positions of dgs , which models the icl formed by acrolein in a cpg sequence context ( 12,16 ) . related icls were constructed between the n - positions of da placing the cross - link in the major groove ( figure 1b and table 1 , rows 3 and 5 ) . an n - da icl1 was designed to model for a potential intermediate of icl processing that could be generated by exo / endonucleolytic removal of dna patch 3 to the icl site . even though n - da icl1 contains an eight nucleotide duplex region 5 to the icl site , it was anticipated that pol  would efficiently catalyze strand displacement dna synthesis due to its intrinsic high strand displacement activity ( 5 ) , a property that is useful for replicating this type of dna lesion . a n - da icl2 was constructed to model another potential intermediate of icl processing ; this could be generated by removal of dna patches both 3 and 5 to the icl site . when these major groove icl - containing dnas were used in primer extension reactions , pol  could extend primers and generate full - length products ( figure 3a , b ) . for both types of icls , the amount of primers extended beyond the adducted site was less as compared to nd substrate ; specifically , a 10- and 4-fold decrease was observed for n - da - icl1 and n - da - icl2 , respectively . as expected , pol  catalyzed strand displacement synthesis on the n - da icl1 template ( figure 3a ) . when the identity of nucleotides incorporated opposite the cross - linked site was examined by single nucleotide incorporations using the n - da icl2 template , the data indicated that pol  preferentially incorporated the correct nucleotide , dt , opposite the n - modified da ( figure 3c ) . replication bypass of n - dan - da interstrand cross - links by human pol . primer extensions were catalyzed by 10 nm human pol  under running start conditions ( 9 primer ) using nd , n - da icl1 ( a ) , or n - da icl2 ( b ) . reactions shown in lanes 12 and lanes 34 were conducted side - by - side , and products were separated on the same gel . ( c ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol  under standing start conditions ( 1 primer ) using nd and n - da icl2 . all reactions were carried out in the presence of 5 nm primer template and 100 m individual or all dntps for 15 min at 37 c . given that pol  was able to bypass the extremely bulky major groove n - da pcls and icls , we tested whether it could replicate past chemically identical pcl and icl lesions except situated in the minor groove through a linkage with the n position of dg ( figure 1c , d ) . primer extensions using these n - dg linked pcls revealed that pol  was strongly blocked one nucleotide prior to both pcls and was completely blocked at the site opposite the lesion , with no further primer extension observed ( figure 4a ) . the identity of the nucleotide(s ) incorporated opposite the pcls was not determined due to the limited nucleotide incorporation . even though pol  could not replicate past the n - dg pcls , suggesting an inability to replicate past minor groove dna adducts , we tested whether pol  could catalyze tls past n - dgn - dg icls ( figure 1d and table 1 , rows 69 ) . no incorporation opposite the lesion site was observed for these icl - containing dnas ( figure 4b ) . thus , similar to the observations made using pcl - containing dnas , pol  has much higher tolerance for the major groove n - da icls than the minor groove n - dg icls . replication bypass of n - dg peptide and interstrand cross - links by human pol . ( a ) primer extensions were catalyzed by 10 nm human pol  under running start conditions ( 4 primer ) in the presence of 5 nm primer template containing nd , n - dg pcl4 , or n - dg pcl12 for 30 min at 37 c . ( b ) primer extensions were catalyzed by 25 nm human pol  under running start conditions ( 10 primer ) in the presence of 7.5 nm primer template containing nd , n - dg icl1 , n - dg icl2 , n - dg icl3 , or n - dg icl4 for 10 min at 37 c . all reactions were carried out in the presence of 100 m dntps . because pol  was able to accurately replicate past major groove n - da pcl and icl lesions , we wanted to further explore the substrate range of pol  and address the question concerning pol  ability to bypass da adducts in which both n1 and n sites are blocked by modification . thus , the ability of pol  to bypass an da ( figure 1e ) was examined . da is a promutagenic lesion that can be formed by exposure to industrial toxicants such as vinyl chloride and by the reaction of dna with bis - electrophiles produced by lipid peroxidation ( 17 ) . the running start primer extensions demonstrated that pol  was severely blocked one nucleotide prior to the da adduct , with only a very small amount of full - length product formed ( figure 5a ) . however , the nucleotide incorporation opposite the lesion appeared to be quite accurate . under the conditions used , the primers were extended in the presence of dttp but not other dntps ( figure 5b ) . on the basis of these data , we conclude that relatively nonbulky modifications at both n1 and n of da are sufficient to inhibit pol -catalyzed tls . replication bypass of 1,n - etheno - da and ( + ) -trans - anti - bpde - da by human pol . ( a ) primer extensions were catalyzed by 10 nm human pol  under running start conditions ( 3 primer ) in the presence of 5 nm primer template containing nd or da . ( b ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol  under standing start conditions ( 1 primer ) in the presence of 5 nm primer template using the same substrates as described in panel a. ( c ) primer extensions were catalyzed by 10 nm human pol  under running start conditions ( 6 primer ) in the presence of 2 nm primer template containing nd or ( + ) -bpde - da . ( d ) single nucleotide incorporations and primer extensions were catalyzed by 5 nm human pol  under standing start conditions ( 1 primer ) in the presence of 2 nm primer template using the same substrates as described in panel c. all reactions were carried out in the presence of 100 m individual or all dntps for 15 min at 37 c . we also investigated the ability of pol  to carry out tls past ( + ) -bpde - da ( figure 1f ) , one of the adducts formed by the active metabolite of the prevalent environmental pollutant , benzo[a]pyrene ( 18 ) . to construct an oligodeoxynucleotide of sufficient length for tls analyses , an 11-mer oligodeoxynucleotide containing the ( + ) -bpde - da was p - labeled , ligated to a 16-mer oligodeoxynucleotide , and gel - purified . residual radioactive signals of the resulting 27-mer template are evident in all lanes [ figure 5c , d , ( + ) -bpde - da-27-mer template ] . primer extensions revealed that pol  could not fully extend the 6 primer and was severely blocked one nucleotide prior to the adducted site ( figure 5c ) . in addition , pol  displayed error - prone patterns of nucleotide incorporation opposite the lesion ( figure 5d ) ; in the presence of datp , 16% of primers were extended , while only 1% of primers were extended in the presence of the correct dttp . a limited primer extension ( < 1% ) was also observed in the presence of dgtp . pol -catalyzed tls past ( + ) -bpde - da was inefficient , incomplete , and error - prone . although very significant progress has been made in both structural analyses of many of the tls pols and the range of damaged dna substrates that these pols can bypass , in the majority of cases , the biological role of these enzymes can only be inferred by changes in efficiency and mutagenic rates of tls in their absence . little data have been gathered concerning the role of these enzymes in developmental biology , their tissue - specific expression and steady - state distribution in adult organisms , the mechanism(s ) for recruitment to sites of dna damage , and the processes that regulate their expressions in response to environmental toxicant exposures and/or endogenous stresses . in the case of pol  , the initial characterizations have demonstrated a very limited repertoire of lesions that pol  can efficiently bypass ( 5 ) . although a cellular role of pol  in tls has not yet been established , its high frequency of misincorporation of dt opposite dg suggests that its expression would be tightly regulated . thus , understanding the potential substrate specificity of pol  may help to guide future investigations of its biological role in tls and circumstances for induction or activation . in this regard , we demonstrated that although pol  could carry out tls past bulky major groove dna lesions such as n - da pcls and n - dan - da icls , it failed to replicate dnas containing chemically similar minor groove dna lesions . thus , the location of the lesion within dna is one important structural determinant that influences the ability of pol  to bypass the lesion . we also discovered that pol  could not efficiently bypass da , and we speculate that this may be either due to pol  requirement for the watsoncrick edge for efficient da templating or structural heterogeneity of the lesion within the active site . regarding the former possibility , pol  may fail to catalyze efficient tls past da because the watsoncrick edge of this adduct is blocked by the modification . the majority of dna pols utilize the geometry of the canonical watsoncrick pairs for the nucleotide selection ( 19 ) , but some tls pols utilize alternative mechanisms . for example , pol  induces a syn conformation on template purines , which results in a hoogsteen base pairing with the correctly matched incoming nucleotide and enables bypass of lesions that disrupt the watsoncrick edge but not the hoogsteen edge ( 20 ) . the diminished capability of pol  to bypass da strongly suggests that the accessibility of the watsoncrick edge of the template da is important for the efficient replication by this pol ; thus , it is likely that pol -catalyzed polymerization involves the recognition of the watsoncrick geometry . with regard to structural heterogeneity of da adduct , nmr analyses have revealed that orientation of the base around the glycosydic bond is affected by identity of nucleotide opposite the lesion ( 21,22 ) . when paired with dt , the da is in a normal anti conformation ; however , the da base prefers the syn conformation when placed opposite a mispaired dg . additionally , the cocrystal structure of dna containing an da bound to pol  also revealed that the lesion adopted a syn conformation ( 20 ) . thus , da can adopt a syn or anti conformation depending on the base it is paired with , as well as interactions with the pol . there is a possibility that similar to pot  , the adducted base is in a syn conformation in the pol  active site . however , it is unclear whether either orientation of the -modified base can be efficiently utilized for nucleotide insertion . intriguingly , pol  also could not fully bypass a ( + ) -bpde - da . the nmr solution structure of this lesion opposite dt in duplex dna showed complex conformational heterogeneity ( 23 ) . computational studies using an nmr structure of duplex dna where dt was placed opposite the modified da have revealed that the glycosydic bond of ( + ) -bpde - da was in syn - anti equilibrium ; therefore , the modified da has a diminished capability to participate in watsoncrick base pairing ( 24 ) . it is possible that the glycosydic bond of modified da rotates and adopts a syn conformation at the primer - template junction and therefore can not participate in watsoncrick base pairing . an alternative explanation may be that the structure of the active site of pol  is significantly distorted by bulky aromatic ring system of this lesion . it has been shown that the bpde moiety of this lesion is intercalated into the double helix , on the 3 side of the adducted da , and thus causes significant structural distortions at the active site of t7 pol ( 25 ) . because the t7 pol is also a member of the a - family pols , the inability of pol  to bypass this lesion may be due to the active site distortions , similar to those shown for t7 pol . with regard to the low fidelity synthesis past this lesion by pol , it has been shown that the adducted da is displaced from the active site of t7 pol as a result of steric hindrance between the bpde moiety and the primer template . among the four dntps , datp fits best into the dntp binding pocket that is enlarged due to the displacement of adducted da ( 25 ) . this structural change provides a likely explanation for the predominant misincorporation of da by pol . recently , we reported a comparative study of the mutagenic consequences of identical lesions linked in the major groove via n - da versus the minor groove linkage via n - dg during the replication of single - stranded pms2 shuttle vectors in cos-7 cells ( 11 ) ; these lesions were the same site - specific pcl4s substrates used in this investigation . these data revealed that dna containing either lesion could be replicated , with the n - dg pcl4 being moderately mutagenic , while the n - da pcl4 showed very low mutagenic potential . although it is anticipated that the extremely bulky nature of the n - da pcls lesions would block replicative pols , we carried out in vitro replication assays using pol  ( figure 6 ) . the primer extensions and single nucleotide incorporations showed some limited bypass of n - da pcl12 at very high dntp concentrations ( 100 m ) ( figure 6a ) , while the titration experiment with varying concentrations of dttp ( from 6.4 nm to 100 m ) revealed the severity of the blockage posed by this lesion for the pol -catalyzed nucleotide insertion ( figure 6b ) . specifically , pol  could incorporate dt opposite the nd da at concentrations as low as 6.4 nm with 8% of primers being extended , while even at 100 m dttp , only 2% of primers were extended on the n - da pcl12-containing substrate . these results suggest that although in vitro conditions could be manipulated to observe bypass of n - da pcls by pol  , the block to replication is likely to occur when pol  encounters such bulky lesions ; therefore , specialized pols may be recruited to carry out tls . the data presented herein suggest that pol  is a likely candidate to perform tls past a variety of major groove lesions . at present , the structural basis is unknown why pol  could efficiently bypass pcl and icl as large as 3025 da but fails to bypass less bulky bpde adduct . however , we speculate that the lesions that are readily bypassed by pol  may have significant conformational flexibility and thus , can be accommodated without distorting dnaenzyme interactions within the pol active site . overall , our data suggest that since pol  is capable of bypassing extremely large major ( but not minor ) groove lesions , it may play a role in tls following toxicant challenges that result in the formation of such lesions in the cells . replication bypass of n - da peptide cross - link by yeast pol . ( a ) single nucleotide incorporations and primer extensions were catalyzed by 0.5 nm yeast pol  with 2 nm primer template containing nd or n - da pcl12 under standing start conditions ( 1 primer ) in the presence or absence of 5 nm pcna for 20 min at 37 c . ( b ) dttp titration assays with 2 nm primer template were catalyzed by 0.5 nm yeast pol  under standing start conditions ( 1 primer ) in the absence of pcna for 10 min at 37 c using the same substrates as described in panel a. dttp concentration ranges from 6.4 nm to 100 m ( 5-fold incremental increase ) .
dna polymerase  ( poln or pol  ) is a newly discovered a family polymerase that generates a high error rate when incorporating nucleotides opposite dg ; its translesion dna synthesis ( tls ) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions . in the current investigation , we describe a novel tls substrate specificity of pol  , demonstrating that it is able to bypass exceptionally large dna lesions whose linkages are through the dna major groove . specifically , pol  catalyzed efficient and high fidelity tls past peptides linked to n6-da via a reduced schiff base linkage with a -hydroxypropano - da . 
 additionally , pol  could bypass dna interstrand cross - links with linkage between n6-das in complementary dna strands . 
 however , the chemically identical dnapeptide and dna interstrand cross - links completely blocked pol  when they were located in the minor groove via a n2-dg linkage . 
 furthermore , we showed that pol  incorporated a nucleotide opposite the 1,n6-etheno - da ( da ) in an error - free manner and ( + ) -trans - anti - benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide - da [ ( + ) -bpde - da ] in an error - prone manner , albeit with a greatly reduced capability . collectively , these data suggest that although pol  bypass capacity can not be generalized to all major groove dna adducts , this polymerase could be involved in tls when genomic replication is blocked by extremely large major groove dna lesions . in view of the recent observation that pol  may have a role in cellular tolerance to dna cross - linking agents , 
 our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some dnaprotein and dnadna cross - links .