Requirement of DNA polymerase eta for error-free bypass of UV-induced CC and TC photoproducts

The yeast RAD30-encoded DNA polymerase eta (Poleta) bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Human DNA polymerase eta functions similarly in the bypass of this lesion, and mutations in human Poleta result in the cancer prone syndrome, the variant form of xeroderma pigmentosum. UV light, however, also elicits the formation of cis-syn cyclobutane dimers and (6-4) photoproducts at 5'-CC-3' and 5'-TC-3' sites, and in both yeast and human DNA, UV-induced mutations occur primarily by 3' C to T transitions. Genetic studies presented here reveal a role for yeast Poleta in the error-free bypass of cyclobutane dimers and (6-4) photoproducts formed at CC and TC sites. Thus, by preventing UV mutagenesis at a wide spectrum of dipyrimidine sites, Poleta plays a pivotal role in minimizing the incidence of sunlight-induced skin cancers in humans.     

Mutagenicity of a unique thymine-thymine dimer or thymine-thymine pyrimidine pyrimidone (6-4) photoproduct in mammalian cells.

The mutagenic properties of UV-induced photoproducts, both the cis-syn thymine-thymine dimer (TT) and the thymine-thymine pyrimidine pyrimidone (6-4) photoproduct [T(6-4)T] were studied in mammalian cells using shuttle vectors. A shuttle vector able to replicate in both mammalian cells and bacteria was produced in its single-stranded DNA form. A unique photoproduct was inserted at a single restriction site and after recircularization of the single-stranded DNA vector, this latter was transfected into simian COS7 cells. After DNA replication the vector was extracted from cells and used to transform bacteria. Amplified DNA was finally analyzed without any selective screening, DNA from randomly picked bacterial colonies being directly sequenced. Our results show clearly that both lesions are mutagenic, but at different levels. Mutation frequencies of 2 and 60% respectively were observed with the TT dimer and the T(6-4)T. With the TT dimer the mutations were targeted on the 3'-T. With the T(6-4)T a large variety of mutations were observed. A majority of G-->T transversions were semi-targeted to the base before the 5'-T of the photoproduct. These kinds of mutations were not observed when the same plasmid was transfected directly into SOS-induced JM105 bacteria or when the T(6-4)T oligonucleotide inserted in a different plasmid was replicated in SOS-induced SMH10 Escherichia coil bacteria. These semi-targeted mutations are therefore the specific result of bypass of the T(6-4)T lesion in COS7 cells by one of the eukaryotic DNA polymerases.     

U-U and T-T cyclobutane dimers have different mutational properties.

We have examined the mutagenic properties in E. coli of single stranded vectors containing a uniquely placed cis-syn or trans-syn uracil-uracil cyclobutane dimer in the sequence 5' GCAAGUUGGAG 3', and compared these with the properties of the corresponding T-T dimers in the same sequence context. The frequencies with which U-U and T-T photoproducts were bypassed were similar in SOS induced cells, and each induced similar frequencies of mutations. However, although both U-U and T-T cis-syn dimers showed a preference for misincorporation in about 5-7% of the replication products, with T or G being incorporated in place of A, the ratios of these events differed, being > 4:1 for T-T cis-syn, but only 2:1 for U-U cis-syn. A shift towards G insertion opposite dimerized uracil was also found with the trans-syn dimers, but the difference was greater; T and G were misincorporated opposite the U-U trans-syn dimer in a ratio of 1:2, compared with 4:1 for its T-T counterpart. In addition, the U-U dimer induced only nucleotide substitutions, unlike the T-T photoproduct which induced single nucleotide deletions as well as substitutions. We conclude that even relatively minor differences in photoproduct structure, such as the presence of a methyl group at C-5, can alter mutational properties, and that such properties cannot depend only on the attributes of the DNA polymerase. Neither the efficiency of bypass, the error frequency nor the mutation spectrum of either U-U isomer is influenced by DNA uracil glycosylase. In vitro, the U-U cis-syn dimer is a substrate for DNA photolyase, but not for the glycosylase.     

Translesion synthesis by yeast DNA polymerase ζ from templates containing lesions of ultraviolet radiation and acetylaminofluorene

In the yeast Saccharomyces cerevisiae, DNA polymerase ζ (Polζ) is required in a major lesion bypass pathway. To help understand the role of Polζ in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polζ performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3′ T, and predominantly A opposite the 5′ T. Purified yeast Polζ predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polζ catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3′ T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3′ T of the TT (6-4) photoproduct. In contrast, the 3′ T of a cis–syn TT dimer completely blocked purified yeast Polζ, whereas the 5′ T was readily bypassed. These results support the following dual-function model of Polζ. First, Polζ catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polζ.     

Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.     

Thermodynamic and base-pairing studies of matched and mismatched DNA dodecamer duplexes containing cis-syn, (6-4) and Dewar photoproducts of TT.

Cis-syn dimers, (6-4) products and their Dewar valence isomers are the major photoproducts of DNA and have different mutagenic properties and rates of repair. To begin to understand the physical basis for these differences, the thermal stability and base pairing properties of the corresponding photoproducts of the TT site in d(GAGTATTATGAG) were investigated. The (6-4) and Dewar products destabilize the duplex form by approximately 6 kcal/mol of free energy at 37 degreesC relative to the parent, whereas a cis-syn dimer only destabilizes the duplex form by 1.5 kcal/mol. Duplexes with G opposite the 3'-T of the (6-4) and Dewar products are more stable than those with A by approximately 0.4 kcal/mol, whereas the cis-syn dimer prefers A over G by 0.7 kcal/mol. Proton NMR suggests that wobble base pairing takes place between the 3'-T of the cis-syn dimer and an opposed G, whereas there is no evidence of significant H-bonding between these two bases in the (6-4) product. The thermodynamic and H-bonding data for the (6-4) product are consistent with a 4 nt interior loop structure which may facilitate flipping of the photoproduct in and out of the helix.     

Reduced efficiency and increased mutagenicity of translesion DNA synthesis across a TT cyclobutane pyrimidine dimer, but not a TT 6-4 photoproduct, in human cells lacking DNA polymerase eta

Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase eta (poleta), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine-thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine-thymine 6-4 photoproduct) in three pairs of poleta-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6-4.4-fold in cells lacking poleta, and exhibited a strong 6-17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in poleta-deficient cells were opposite the 3'T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of poleta, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in poleta-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27-63%), with multiple mutations types, and no significant difference between cells with or without poleta. Approximately 50% of the mutations formed were semi-targeted, of which 84-93% were due to the insertion of an A opposite the template G 5' to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of poleta in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of poleta in TLS across 6-4 PP under some conditions.     

Utilization of DNA photolyase, pyrimidine dimer endonucleases, and alkali hydrolysis in the analysis of aberrant ABC excinuclease incisions adjacent to UV-induced DNA photoproducts.

ABC excinuclease of Escherichia coli removes 6-4 photoproducts and pyrimidine dimers from DNA by making two single strand incisions, one 8 phosphodiester bonds 5' and another 4 or 5 phosphodiester bonds 3' to the lesion. We describe in this communication a method, which utilizes DNA photolyase from E. coli, pyrimidine dimer endonucleases from M. luteus and bacteriophage T4, and alkali hydrolysis, for analyzing the ABC excinuclease incision pattern corresponding to each of these photoproducts in a DNA fragment. On occasion, ABC excinuclease does not incise DNA exclusively 8 phosphodiester bonds 5' or 4 or 5 phosphodiester bonds 3' to the photoproduct. Both the nature of the adduct (6-4 photoproduct or pyrimidine dimer) and the sequence of neighboring nucleotides influence the incision pattern of ABC excinuclease. We show directly that photolyase stimulates the removal of pyrimidine dimers (but not 6-4 photoproducts) by the excinuclease. Also, photolyase does not repair CC pyrimidine dimers efficiently while it does repair TT or TC pyrimidine dimers.     

Mismatch extension ability of yeast and human DNA polymerase eta

DNA polymerase eta (Poleta) functions in error-free replication of UV-damaged DNA, and in vitro it efficiently bypasses a cis-syn T-T dimer by incorporating two adenines opposite the lesion. Steady state kinetic studies have shown that both yeast and human Poleta are low-fidelity enzymes, and they misincorporate nucleotides with a frequency of 10(-2)-10(-3) on both undamaged and T-T dimer-containing DNA templates. To better understand the role of Poleta in error-free translesion DNA synthesis, here we examine the ability of Poleta to extend from base mismatches. We find that both yeast and human Poleta extend from mismatched base pairs with a frequency of approximately 10(-3) relative to matched base pairs. In the absence of efficient extension of mismatched primer termini, the ensuing dissociation of Poleta from DNA may favor the excision of mismatched nucleotides by a proofreading exonuclease. Thus, we expect DNA synthesis by Poleta to be more accurate than that predicted from the fidelity of nucleotide incorporation alone.     

The (6-4) photoproduct of thymine-thymine induces targeted substitution mutations in mammalian cells.

Two major ultraviolet-induced photolesions of TpT, a (6-4) photoproduct [T(6-4)T] and a cis-syn cyclobutane TT dimer (T=T), were incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the modified DNAs were transfected into simian COS-7 cells. The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli. The DNA replication efficiencies of plasmids containing T(6-4)T and T=T in the template strand for lagging strand synthesis were 93 and 79%, respectively, as compared with the unmodified DNA. Similar inhibitory effects were observed in the cases of the photoproducts in the template strand for leading strand synthesis (71 and 58%, respectively). These results indicated that T(6-4)T blocked DNA replication more weakly than T=T during leading and lagging strand syntheses in mammalian cells. The mutation frequencies of T(6-4)T were 2.3 and 4.7% in the leading and lagging template strands, respectively. The T=T lesion was less mutagenic and induced mutations with 0.2-0.7% frequencies. The T(6-4)T lesion primarily elicited 3'-T-->C substitutions, and T=T induced various types of mutations. These results indicate that T(6-4)T is more mutagenic than T=T during leading and lagging strand syntheses in simian cells. Moreover, this is the first evidence that shows T(6-4)T mainly elicits targeted substitutions at its 3'-T site in mammalian cells.     

Replication past O(6)-methylguanine by yeast and human DNA polymerase eta

O(6)-Methylguanine (m6G) is formed by the action of alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on DNA. m6G is a highly mutagenic and carcinogenic lesion, and it presents a block to synthesis by DNA polymerases. Here, we provide genetic and biochemical evidence for the involvement of yeast and human DNA polymerase eta (Poleta) in the replicative bypass of m6G lesions in DNA. The formation of MNNG-induced mutations is almost abolished in the rad30Delta pol32Delta double mutant of yeast, which lacks the RAD30 gene that encodes Poleta and the Pol32 subunit of DNA polymerase delta (Poldelta). Although Poldelta can function in the mutagenic bypass of m6G lesions, our biochemical studies indicate that Poleta is much more efficient in replicating through m6G than Poldelta. Both Poleta and Poldelta insert a C or a T residue opposite from m6G; Poleta, however, is more accurate, as it inserts a C about twice as frequently as Poldelta. Alkylating agents are used in the treatment of malignant tumors, including lymphomas, brain tumors, melanomas, and gastrointestinal carcinomas, and the clinical effectiveness of these agents derives at least in part from their ability to form m6G in DNA. Inactivation of Poleta could afford a useful strategy for enhancing the effectiveness of these agents in cancer chemotherapy.     

Use of a combined enzymatic digestion/ESI mass spectrometry assay to study the effect of TATA-binding protein on photoproduct formation in a TATA box

Recently, it was reported that TATA-binding protein (TBP) enhances (6-4) photoproduct formation in a TATA box under UVC irradiation [Aboussekhra and Thoma (1999) EMBO J. 18, 433-443]. The conclusions of that study were based on an indirect enzymatic assay that was not specific for (6-4) photoproducts. Herein we report the use of a recently developed coupled enzymatic digestion/mass spectrometry assay [Wang et al. (1999) Chem. Res. Toxicol. 12, 1077-1082] to identify unambiguously and quantify the photoproducts formed in a TATA box-containing dodecamer duplex sequence in the presence or absence of TBP binding. Exposure of the adenovirus major late promoter TATA box to a high dose of UVC irradiation in the absence of the C-terminal domain of yeast TBP leads to predominant formation of the cis-syn dimer within the T(4) tract, whereas exposure in the presence of TBP leads to almost exclusive formation of the (6-4) photoproduct. In contrast, the (6-4) product is not detected at high doses of UVB irradiation in the absence of TBP but is detected in the presence of TBP, although the cis-syn product predominates. When the products of UVB irradiation were subsequently exposed to a high dose of UVC irradiation in the presence of TBP, the (6-4) photoproduct again becomes nearly the exclusive photoproduct, indicating that the cis-syn dimer is being reversed to TT by UVC light. Both cis-syn and (6-4) photoproducts are formed in approximately equal amounts upon irradiation with small doses of UVC in the presence of TBP, but the fraction of (6-4) photoproduct increases with dose. Through the use of a TATA box containing a site-specifically deuterated thymine, it was found that (6-4) photoproducts formed most selectively at the second and third positions of the T(4) tract upon either UVB or UVC irradiation in the presence of TBP. By using the same substrate, it was found that UVC-induced TA formation was inhibited by TBP binding and that TA formation was greatest at the 5' end of the TATA sequence.     

Misinsertion and bypass of thymine-thymine dimers by human DNA polymerase iota

Human DNA polymerase iota (pol(iota)) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that poliota is able to facilitate limited translesion replication of a thymine-thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that pol(iota) misinserts T or G opposite the 3' T of the CPD approximately 1.5 times more frequently than the correct base, A. While pol(iota) is unable to extend the T.T mispair significantly, the G.T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A. T base pair. By comparison, pol(iota) readily misinserts two bases opposite a 6-4 thymine-thymine pyrimidine-pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that poliota possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.     

Two-step error-prone bypass of the (+)- and (-)-trans-anti-BPDE-N2-dG adducts by human DNA polymerases eta and kappa

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon (PAH) associated with potent carcinogenic activity. Mutagenesis induced by benzo[a]pyrene DNA adducts is believed to involve error-prone translesion synthesis opposite the lesion. However, the DNA polymerase involved in this process has not been clearly defined in eukaryotes. Here, we provide biochemical evidence suggesting a role for DNA polymerase eta (Poleta) in mutagenesis induced by benzo[a]pyrene DNA adducts in cells. Purified human Poleta predominantly inserted an A opposite a template (+)- and (-)-trans-anti-BPDE-N2-dG, two important DNA adducts of benzo[a]pyrene. Both lesions also dramatically elevated G and T mis-insertion error rates of human Poleta. Error-prone nucleotide insertion by human Poleta was more efficient opposite the (+)-trans-anti-BPDE-N2-dG adduct than opposite the (-)-trans-anti-BPDE-N2-dG. However, translesion synthesis by human Poleta largely stopped opposite the lesion and at one nucleotide downstream of the lesion (+1 extension). The limited extension synthesis of human Poleta from opposite the lesion was strongly affected by the stereochemistry of the trans-anti-BPDE-N2-dG adducts, the nucleotide opposite the lesion, and the sequence context 5' to the lesion. By combining the nucleotide insertion activity of human Poleta and the extension synthesis activity of human Polkappa, effective error-prone lesion bypass was achieved in vitro in response to the (+)- and (-)-trans-anti-BPDE-N2-dG DNA adducts.     

Solid phase-supported thymine dimers for the construction of dimer-containing DNA by combined chemical and enzymatic synthesis: a potentially general method for the efficient incorporation of modified nucleotides into DNA.

The ability to study the structure-activity relationships of the cis-syn thymine dimer, the major photoproduct of DNA, has been greatly aided by the availability of a building block suitable for its sequence-specific incorporation into oligonucleotides by standard automated DNA synthesis. Unfortunately, its usefulness is compromised by the fact that it takes six steps to synthesize in low overall yield and, as with all phosphoramidite building blocks, has to be used in great excess over the support in standard automated synthesis. To extend the usefulness of this building block, we have directly coupled it to standard A, C, G and T long chain alkylamine-linked controlled pore glass supports to yield a solid phase-supported dimer. We then demonstrate that 13mers containing a 3'-terminal d(T[cis-syn]TN) group synthesized with this support at 0.2 micromol scale can be efficiently incorporated into longer oligonucleotides by both primer extension with 3'-->5'exonuclease-deficient Klenow fragment or T4 polymerase and dNTPs or by enzymatic ligation with T4 DNA ligase to another oligonucleotide opposite a complementary template. The site specificity and integrity of the cis-syn thymine dimer after both primer extension and ligation was confirmed by cis-syn dimer-specific cleavage with T4 denV endonuclease V. This general approach should be applicable to the synthesis of many types of site-specific nucleic acid modifications and would be of particular use for those for which the required building blocks are expensive or difficult to make.     

DNA polymerase theta (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

DNA polymerase theta (pol theta) is a nuclear A-family DNA polymerase encoded by the POLQ gene in vertebrate cells. The biochemical properties of pol theta and of Polq-defective mice have suggested that pol theta participates in DNA damage tolerance. For example, pol theta was previously found to be proficient not only in incorporation of a nucleotide opposite a thymine glycol or an abasic site, but also extends a polynucleotide chain efficiently from the base opposite the lesion. We carried out experiments to determine whether this ability to extend from non-standard termini is a more general property of the enzyme. Pol theta extended relatively efficiently from matched termini as well as termini with A:G, A:T and A:C mismatches, with less descrimination than a well-studied A-family DNA polymerase, exonuclease-free pol I from E. coli. Although pol theta was unable to, by itself, bypass a cyclobutane pyrimidine dimer or a (6-4) photoproduct, it could perform some extension from primers with bases placed across from these lesions. When pol theta was combined with DNA polymerase iota, an enzyme that can insert a base opposite a UV-induced (6-4) photoproduct, complete bypass of a (6-4) photoproduct was possible. These data show that in addition to its ability to insert nucleotides opposite some DNA lesions, pol theta is proficient at extension of unpaired termini. These results show the potential of pol theta to act as an extender after incorporation of nucleotides by other DNA polymerases, and aid in understanding the role of pol theta in somatic mutagenesis and genome instability.     

Specific binding of human MSH2.MSH6 mismatch-repair protein heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.

Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007-1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T cis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible bases opposite the respective 3'-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2.MSH6 heterodimer protein (hMutSalpha). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSalpha affinity than photoproduct/AA "matches"; the apparent affinity of hMutSalpha for the ((6-4)photoproduct)/AA-"matched" substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSalpha affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSalpha to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium).     

Error-free and error-prone lesion bypass by human DNA polymerase kappa in vitro

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polκ encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polκ is a novel lesion bypass polymerase in vitro. Purified human Polκ efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polκ most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polκ was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polκ effectively bypassed a template (–)-trans-anti-benzo[a]pyrene-N²-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polκ was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polκ plays an important role in both error-free and error-prone lesion bypass in humans.     

In vitro evidence that UV-induced frameshift and substitution mutations at T tracts are the result of misalignment-mediated replication past a specific thymine dimer.

A previous study of UV-induced (254 nm) mutations in the lacI gene of Escherichia coli found that frameshift mutations accounted for about 35% of the observed mutations and that these mutations occurred predominantly at An.Tn sequences [Miller, J.H. (1985) J. Mol. Biol. 182, 48-65]. Because An.Tn sequences are hotspots for cis-syn thymine dimer formation [Brash, D.E., & Haseltine, W. A. (1982) Nature 298, 189-192], it would appear that UV-induced frameshift mutations are the result of an error during replicative bypass of a thymine dimer within such a sequence. To test the validity of such a proposal, replication experiments were carried out on templates containing cis-syn thymine dimers at each of the five possible sites of a T6 tract. The 59-mer templates were prepared by ligating oligonucleotides containing an EcoRI site to the 5'-end of decamers containing the cis-syn thymine dimer and oligonucleotides containing the primer site to the 3'-end. Primer-extension reactions were then carried out on these templates with a 3'----5' exonuclease-deficient (exo-) Klenow fragment of E. coli polymerase I and an exo-T7 polymerase (Sequenase Version 2.0). The replicative bypass products were cleaved with EcoRI to rigorously establish and quantify the presence of frameshift mutations. Both polymerases were able to bypass dimers at all sites, but only the exo-T7 polymerase led to detectable frameshifts, both -1 (approximately 30%) and -2 (approximately 5%), and only with the template containing a cyclobutane dimer at the second site from the 5'-end of the T6 tract. Sequencing of the T7 polymerase-catalyzed bypass products of all templates demonstrated that within the limits of discrimination only As were introduced opposite the dimer-containing T tracts. The only exception was for the template with the dimer at the second site which led to a readily detectable amount of a substitution mutation (approximately 30%) opposite the 5'-thymine of the T6 tract. A mechanism involving a competition between reversible misalignment and realignment steps and irreversible elongation steps is proposed to explain the origin of both the frameshift and the substitution mutations. The implications of this work to the mechanism of UV-induced frameshift and substitution mutations at T tracts in vivo are discussed.     

The 3' --> 5' exonuclease of T4 DNA polymerase removes premutagenic alkyl mispairs and contributes to futile cycling at O6-methylguanine lesions

We have studied the processing of O(6)-methylguanine (m6G)-containing oligonucleotides and N-methyl-N-nitrosourea (MNU)-treated DNA templates by the 3' --> 5' exonuclease of T4 DNA polymerase. In vitro biochemical analyses demonstrate that the exonuclease can remove bases opposite a defined m6G lesion. The efficiency of excision of a terminal m6G.T was similar to that of m6G.C, and both were excised as efficiently as a G.T substrate. Partitioning assays between the polymerase and exonuclease activities, performed in the presence of dNTPs, resulted in repeated incorporation and excision events opposite the m6G lesion. This idling produces dramatically less full-length product, relative to natural substrates, indicating that the 3' --> 5' exonuclease may contribute to DNA synthesis inhibition by alkylating agents. Genetic data obtained using an in vitro herpes simplex virus-thymidine kinase assay support the inefficiency of the exonuclease as a "proofreading" activity for m6G, since virtually all mutations produced by the native enzyme using MNU-treated templates were G --> A transitions. Comparison of MNU dose-response curves for exonuclease-proficient and -deficient forms of T4 polymerase reveals that the exonuclease efficiently removes 50-86% of total premutagenic alkyl mispairs. We propose that idling of exonuclease-proficient polymerases at m6G lesions during repair DNA synthesis provides the biochemical explanation for cellular cytotoxicity of methylating agents.