In vitro effects of a C4'-oxidized abasic site on DNA polymerases

Oxidative damage to DNA produces abasic sites resulting from the formal hydrolysis of the nucleotides' glycosidic bonds, along with a variety of oxidized abasic sites. The C4'-oxidized abasic site (C4-AP) is produced by several DNA-damaging agents. This lesion accounts for approximately 40% of the DNA damage produced by bleomycin. The effect of a C4'-oxidized abasic site incorporated at a defined site in a template was examined on Klenow fragments with and without 3' --> 5' exonuclease activity. Both enzymes preferentially incorporated dA > dG > dC, T opposite C4-AP. Neither enzyme is able to extend the primer past the lesion. Experiments with regular AP sites in an otherwise identical template indicate that Klenow does not differentiate between these two disparate abasic sites. Extension of the primer by alternative polymerases pol II, pol II exo(-), pol IV, and pol V was examined. Pol II exo(-) was most efficient. Qualitative translesion synthesis experiments showed that pol II exo(-) preferentially incorporates T opposite C4-AP, followed in order by dG, dA, and dC. Thymidine incorporation opposite C4'-AP is distinct from the pol II exonuclease interaction with a regular AP site in an otherwise identical template. These in vitro experiments suggest that bypass polymerases may play a crucial role in survival of cells in which C4-AP is produced, and unlike a typical AP site, the C4-AP lesion may not follow the "A-rule". The interaction between bypass polymerases and a C4-AP lesion could explain the high levels of G:C --> T:A transversions in cells treated with bleomycin.     

Repair of oxidized abasic sites by exonuclease III, endonuclease IV, and endonuclease III

2-Deoxyribonolactone (L) and the C4'-oxidized abasic site (C4-AP) are produced by a variety of DNA-damaging agents. If not repaired, these lesions can be mutagenic. Exonuclease III and endonuclease IV are the major enzymes in E. coli responsible for 5'-incision of abasic sites (APs), the first steps in AP repair. Endonuclease III efficiently excises AP lesions via intermediate Schiff-base formation. Incision of L and C4-AP lesions by exonuclease III and endonuclease IV was determined under steady-state conditions using oligonucleotide duplexes containing the lesions at defined sites. An abasic lesion (AP) in an otherwise identical DNA sequence was incised by exonuclease III or endonuclease IV approximately 6-fold more efficiently than either of the oxidized abasic sites (L, C4-AP). Endonuclease IV incision efficiency of 2-deoxyribonolactone or C4-AP was independent of whether the lesion was opposite dA or dG. 2-Deoxyribonolactone is known to cross-link to endonuclease III (Hashimoto, M. (2001) J. Am. Chem. Soc. 123, 3161.). However, the C4-AP lesion is efficiently excised by endonuclease III. Oxidized abasic site repair by endonuclease IV and endonuclease III (C4-AP only) is approximately 100-fold less efficient than repair by exonuclease III. These results suggest that the first step of C4-AP and L oxidized abasic site repair will be the same as that of regular AP lesions in E. coli.     

Probing DNA interstrand cross-link formation by an oxidized abasic site using nonnative nucleotides

The C4'-oxidized abasic site (C4-AP) forms two types of interstrand cross-links with the adjacent nucleotides in DNA. Previous experiments revealed that dG does not react with the lesion and that formation of one type of cross-link is catalyzed by the opposing dA. iso-Guanosine·dC and 2-aminopurine·dT base pairs were used to determine why dG does not cross-link with C4-AP despite its well known reactivity with other bis-electrophiles. 7-Deaza-2'-deoxyadenosine was used to probe the role of the nucleotide opposite C4-AP in the catalysis of interstrand cross-link formation.     

Mechanisms of accurate translesion synthesis by human DNA polymerase eta

The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta (pol eta), which is involved in the replication of damaged DNA. Pol eta catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol eta can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol eta preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol eta could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol eta could continue chain elongation, whereas pol alpha could not. Thus, the fidelity of translesion synthesis by human pol eta relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion.     

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast.     

Analysis of unassisted translesion replication by the DNA polymerase III holoenzyme

DNA damage-induced mutations are formed when damaged nucleotides present in single-stranded DNA are replicated. We have developed a new method for the preparation of gapped plasmids containing site-specific damaged nucleotides, as model DNA substrates for translesion replication. Using these substrates, we show that the DNA polymerase III holoenzyme from Escherichia coli can bypass a synthetic abasic site analogue with high efficiency (30% bypass in 16 min), unassisted by other proteins. The theta and tau subunits of the polymerase were not essential for bypass. No bypass was observed when the enzyme was assayed on a synthetic 60-mer oligonucleotide carrying the same lesion, and bypass on a linear gapped plasmid was 3-4-fold slower than on a circular gapped plasmid. There was no difference in the bypass when standing-start and running-start replication were compared. A comparison of translesion replication by DNA polymerase I, DNA polymerase II, the DNA polymerase III core, and the DNA polymerase III holoenzyme clearly showed that the DNA polymerase III holoenzyme was by far the most effective in performing translesion replication. This was not only due to the high processivity of the pol III holoenzyme, because increasing the processivity of pol II by adding the gamma complex and beta subunit, did not increase bypass. These results support the model that SOS regulation was imposed on a fundamentally constitutive translesion replication reaction to achieve tight control of mutagenesis.     

Solution structure of the N-(deoxyguanosin-8-yl)-1-aminopyrene ([AP]dG) adduct opposite dA in a DNA duplex.

Solution structural studies have been undertaken on the aminopyrene-C(8)-dG ([AP]dG) adduct in the d(C5-[AP]G6-C7). d(G16-A17-G18) sequence context in an 11-mer duplex with dA opposite [AP]dG, using proton-proton distance and intensity restraints derived from NMR data in combination with distance-restrained molecular mechanics and intensity-restrained relaxation matrix refinement calculations. The exchangeable and nonexchangeable protons of the aminopyrene and the nucleic acid were assigned following analysis of two-dimensional NMR data sets on the [AP]dG.dA 11-mer duplex in H2O and D2O solution. The broadening of several resonances within the d(G16-A17-G18) segment positioned opposite the [AP]dG6 lesion site resulted in weaker NOEs, involving these protons in the adduct duplex. Both proton and carbon NMR data are consistent with a syn glycosidic torsion angle for the [AP]dG6 residue in the adduct duplex. The aminopyrene ring of [AP]dG6 is intercalated into the DNA helix between intact Watson-Crick dC5.dG18 and dC7.dG16 base pairs and is in contact with dC5, dC7, dG16, dA17, and dG18 residues that form a hydrophobic pocket around it. The intercalated AP ring of [AP]dG6 stacks over the purine ring of dG16 and, to a lesser extent dG18, while the looped out deoxyguanosine ring of [AP]dG6 stacks over dC5 in the solution structure of the adduct duplex. The dA17 base opposite the adduct site is not looped out of the helix but rather participates in an in-plane platform with adjacent dG18 in some of the refined structures of the adduct duplex. The solution structures are quite different for the [AP]dG.dA 11-mer duplex containing the larger aminopyrene ring (reported in this study) relative to the previously published [AF]dG.dA 11-mer duplex containing the smaller aminofluorene ring (Norman et al., Biochemistry 28, 7462-7476, 1989) in the same sequence context. Both the modified syn guanine and the dA positioned opposite it are stacked into the helix with the aminofluorene chromophore displaced into the minor groove in the latter adduct duplex. By contrast, the aminopyrenyl ring participates in an intercalated base-displaced structure in the present study of the [AP]dG.dA 11-mer duplex and in a previously published study of the [AP]dG.dC 11-mer duplex (Mao et al., Biochemistry 35, 12659-12670, 1996). Such intercalated base-displaced structures without hydrogen bonding between the [AP]dG adduct and dC or mismatched dA residues positioned opposite it, if present at a replication fork, may cause polymerase stalling and formation of a slipped intermediate that could produce frameshift mutations, the most dominant mutagenic consequence of the [AP]dG lesion.     

Translesion synthesis across abasic lesions by human B-family and Y-family DNA polymerases α, δ, η, ι, κ, and REV1

Abasic (apurinic/apyrimidinic, AP) sites are the most common DNA lesions formed in cells, induce severe blocks to DNA replication, and are highly mutagenic. Human Y-family translesion DNA polymerases (pols) such as pols η, ι, κ, and REV1 have been suggested to play roles in replicative bypass across many DNA lesions where B-family replicative pols stall, but their individual catalytic functions in AP site bypass are not well understood. In this study, oligonucleotides containing a synthetic abasic lesion (tetrahydrofuran analogue) were compared for catalytic efficiency and base selectivity with human Y-family pols η, ι, κ, and REV1 and B-family pols α and δ. Pol η and pol δ/proliferating cell nuclear antigen (PCNA) copied past AP sites quite effectively and generated products ranging from one-base to full-length extension. Pol ι and REV1 readily incorporated one base opposite AP sites but then stopped. Pols κ and α were severely blocked at AP sites. Pol η preferentially inserted T and A; pol ι inserted T, G, and A; pol κ inserted C and A; REV1 preferentially inserted C opposite AP sites. The B-family pols α and δ/PCNA preferentially inserted A (85% and 58%, respectively) consonant with the A-rule hypothesis. Pols η and δ/PCNA were much more efficient in next-base extension, preferably from A positioned opposite an AP site, than pol κ. These results suggest that AP sites might be bypassed with moderate efficiency by single B- and Y-family pols or combinations, possibly by REV1 and pols ι, η, and δ/PCNA at the insertion step opposite the lesion and by pols η and δ/PCNA at the subsequent extension step. The patterns of the base preferences of human B-family and Y-family pols in both insertion and extension are pertinent to some of the mutagenesis events induced by AP lesions in human cells.     

Translesion replication by DNA polymerase delta depends on processivity accessory proteins and differs in specificity from DNA polymerase beta

Mutations caused by DNA damage lead to the development of cancer. The critical step in the formation of these mutations is the replication of unrepaired lesions in DNA by DNA polymerases, a process termed translesion replication. Using a newly developed method for preparation of gapped plasmids, containing a site-specific synthetic abasic site, we analyzed translesion replication with purified mammalian DNA polymerases delta and beta. DNA polymerase delta was found to be unable to replicate through the abasic site. Addition of the sliding DNA clamp PCNA, the clamp loader RFC, and ATP caused a drastic 30-fold increase in translesion replication. Thus, similar to Escherichia coli DNA polymerase III, the processivity accessory proteins enable DNA polymerase delta to bypass blocking lesions. Under comparable conditions, DNA polymerase beta was unable to bypass the abasic site, unless its concentration was greatly increased. Analysis of translesion replication products revealed a marked difference in the specificity of bypass: whereas 90% of bypass events by DNA polymerase delta holoenzyme involved insertion of a dAMP residue opposite the abasic site, DNA polymerase beta tended to skip over the abasic site, producing mainly minus frameshifts (73%). The significance of these results for in vivo translesion replication is discussed.     

Mechanisms by which herpes simplex virus DNA polymerase limits translesion synthesis through abasic sites

Results suggest a high probability that abasic (AP) sites occur at least once per herpes simplex virus type 1 (HSV-1) genome. The parameters that control the ability of HSV-1 DNA polymerase (pol) to engage in AP translesion synthesis (TLS) were examined because AP lesions could influence the completion and fidelity of viral DNA synthesis. Pre-steady-state kinetic experiments demonstrated that wildtype (WT) and exonuclease-deficient (exo-) pol could incorporate opposite an AP lesion, but full TLS required absence of exo function. Virtually all of the WT pol was bound at the exo site to AP-containing primer-templates (P/Ts) at equilibrium, and the pre-steady-state rate of excision by WT pol was higher on AP-containing than on matched DNA. However, several factors influencing polymerization work synergistically with exo activity to prevent HSV-1 pol from engaging in TLS. Although the pre-steady-state catalytic rate constant for insertion of dATP opposite a T or AP site was similar, ground-state-binding affinity of dATP for insertion opposite an AP site was reduced 3-9-fold. Single-turnover running-start experiments demonstrated a reduced proportion of P/Ts extended to the AP site compared to the preceding site during processive synthesis by WT or exo- pol. Only the exo- pol engaged in TLS, though inefficiently and without burst kinetics, suggesting a much slower rate-limiting step for extension beyond the AP site.     

Effects of the C4'-oxidized abasic site on replication in Escherichia coli. An unusually large deletion is induced by a small lesion

The C4'-oxidized abasic site (C4-AP) is produced in DNA as a result of oxidative stress by a variety of agents. For instance, the lesion accounts for approximately 40% of the DNA damage produced by the antitumor antibiotic bleomycin. The effect of C4-AP on DNA replication in Escherichia coli was determined using the restriction endonuclease and postlabeling (REAP) method. Three-nucleotide deletion products are the sole products observed following replication of plasmids containing C4-AP under SOS conditions in wild-type cells. Full-length products are formed in varying amounts depending upon the local sequence in wild-type cells under non-SOS-induced conditions. The "A-rule" is followed for the formation of substitution products. C4-AP is the first example of a DNA lesion that produces significant levels of three-nucleotide deletions in a variety of sequence contexts. Experiments carried out in cells lacking specific polymerases reveal that formation of three-nucleotide deletion products results from a coordinated effort involving pol II and pol IV. This is the first example in which these SOS inducible polymerases are shown to work in concert during lesion bypass. Three-nucleotide deletions are not observed during the replication of other abasic lesions, and are rarely produced by bulky adducts. The effect of C4-AP on DNA replication suggests a significant role for this lesion in the cytotoxicity of bleomycin. Formation of the C4-AP lesion may also be responsible for the formation of mutant proteins containing single-amino acid deletions that exhibit altered phenotypes.     

Lesion bypass by the Escherichia coli DNA polymerase V requires assembly of a RecA nucleoprotein filament

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgammaS, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.     

In vitro replication and repair of DNA containing a C2'-oxidized abasic site

Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides. Nonetheless, polymerase and repair enzymes distinguish between various oxidized abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases and repair enzymes are unknown. A recently reported method for the chemical synthesis of oligonucleotides containing C2-AP at a defined site was utilized for studying the activity of Klenow exo(-) and repair enzymes on templates containing the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but extension of the primer past the lesion is strongly blocked. C2-AP is incised less efficiently by exonuclease III and endonuclease IV than are other abasic lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III can be chemically trapped, the location of the 3'-phosphate alpha with respect to the aldehyde prevents beta-elimination associated with the lyase activity of type I base excision repair enzymes. The interactions of the C2'-oxidized abasic site with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic and that it will be removed by strand displacement synthesis and flap endonuclease processing via a long patch repair mechanism.     

Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI).

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.     

Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine

Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N²-deoxyguanosine (dG) and N⁶-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol γ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol γ opposite the BaP DE–dG adducts resembles that observed with the Y family pol η. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.     

Human DNA polymerases lambda and beta show different efficiencies of translesion DNA synthesis past abasic sites and alternative mechanisms for frameshift generation

Human DNA polymerases (pols) beta and lambda could promote template slippage and generate -1 frameshifts on defined heteropolymeric DNA substrates containing a single abasic site. Kinetic data demonstrated that pol lambda was more efficient than pol beta in catalyzing translesion DNA synthesis past an abasic site, particularly in the presence of low nucleotide concentrations. Moreover, pol lambda was found to generate frameshifts in two ways: first, by using a nucleotide-stabilized primer misalignment mechanism, or second, by promoting primer reannealing using microhomology regions between the terminal primer sequence and the template strand. Our results suggest a molecular mechanism for the observed high in vivo rate of frameshifts generation by pol lambda and highlight the remarkable ability of pol lambda to promote microhomology pairing between two DNA strands, further supporting its proposed role in the nonhomologous end joining process.     

In vitro gap-directed translesion DNA synthesis of an abasic site involving human DNA polymerases epsilon, lambda, and beta

DNA polymerase (pol) ε is thought to be the leading strand replicase in eukaryotes, whereas pols λ and β are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols λ, β, and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε, likely by initiating the 3'OHs created at the lesion by the arrested pol ε. However, in the case of pols λ and β, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ε, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol η. Characterization of the reaction products showed that pol β inserted dAMP opposite the AP site, whereas gap filling synthesis by pol λ resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ε and the subsequent TLS by pols λ and β are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol λ at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ε and the repair pols λ and β.     

Lesion (in)tolerance reveals insights into DNA replication fidelity

The initial encounter of an unrepaired DNA lesion is likely to be with a replicative DNA polymerase, and the outcome of this event determines whether an error-prone or error-free damage avoidance pathway is taken. To understand the atomic details of this critical encounter, we have determined the crystal structures of the pol alpha family RB69 DNA polymerase with DNA containing the two most prevalent, spontaneously generated premutagenic lesions, an abasic site and 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG). Identification of the interactions between these damaged nucleotides and the active site provides insight into the capacity of the polymerase to incorporate a base opposite the lesion. A novel open, catalytically inactive conformation of the DNA polymerase has been identified in the complex with a primed abasic site template. This structure provides the first molecular characterization of the DNA synthesis barrier caused by an abasic site and suggests a general mechanism for polymerase fidelity. In contrast, the structure of the ternary 8-oxodG:dCTP complex is almost identical to the replicating complex containing unmodified DNA, explaining the relative ease and fidelity by which this lesion is bypassed.