Processing of bistranded abasic DNA clusters in gamma-irradiated human hematopoietic cells

Clustered DNA damages—two or more lesions on opposing strands and within one or two helical turns—are formed in cells by ionizing radiation or radiomimetic antitumor drugs. They are hypothesized to be difficult to repair, and thus are critical biological damages. Since individual abasic sites can be cytotoxic or mutagenic, abasic DNA clusters are likely to have significant cellular impact. Using a novel approach for distinguishing abasic clusters that are very closely spaced (putrescine cleavage) or less closely spaced (Nfo protein cleavage), we measured induction and processing of abasic clusters in 28SC human monocytes that were exposed to ionizing radiation. γ-rays induced ~1 double-strand break: 1.3 putrescine-detected abasic clusters: 0.8 Nfo-detected abasic clusters. After irradiation, the 28SC cells rejoined double-strand breaks efficiently within 24 h. In contrast, in these cells, the levels of abasic clusters decreased very slowly over 14 days to background levels. In vitro repair experiments that used 28SC cell extracts further support the idea of slow processing of specific, closely spaced abasic clusters. Although some clusters were removed by active cellular repair, a substantial number was apparently decreased by ‘splitting’ during DNA replication and subsequent cell division. The existence of abasic clusters in 28SC monocytes, several days after irradiation suggests that they constitute persistent damages that could lead to mutation or cell killing.     

The chemical stability of abasic RNA compared to abasic DNA

We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2′-O-protecting group together with the analogous DNA and the 2′-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2′-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37°C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via β,δ-elimination and 2′,3′-cyclophosphate formation. We found that β,δ-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only β,δ-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Solution structure of an oligonucleotide containing an abasic site: evidence for an unusual deoxyribose conformation

The antitumor antibiotic bleomycin causes two major lesions in the deoxyribose backbone of DNA: formation of 4′-keto abasic sites and formation of strand breaks with 3′-phosphoglycolate and 5′-phosphate ends. As a model for the 4′-keto abasic site, we have characterized an abasic site (X) in d(CCAAAGXACTGGG)·d(CCCAGTACTTTGG) by two-dimensional NMR spectroscopy. A total of 475 NOEs and 101 dihedral angles provided the restraints for molecular modeling. Four unusual NOEs were observed between each anomer of the abasic site and the neighboring bases. In addition, four coupling constants for adjacent protons of the deoxyribose of both the α and β anomers of the abasic site were observed. The modeling suggests that for both anomers the abasic site is extrahelical, without significant distortion of the backbone opposite the lesion. The coupling constants further allowed assignment of an unusual sugar pucker for each anomer. The unique position of the abasic site in our structural model for each anomer is discussed in terms of repair of such lesions in vivo.     

Endogenous DNA damage clusters in human hematopoietic stem and progenitor cells

Clustered DNA damages-multiple oxidized bases, abasic sites, or strand breaks within a few helical turns-are potentially mutagenic and lethal alterations induced by ionizing radiation. Endogenous clusters are found at low frequencies in unirradiated normal human cells and tissues. Radiation-sensitive hematopoietic cells with low glycosylase levels (TK6 and WI-L2-NS) accumulate oxidized base clusters but not abasic clusters, indicating that cellular repair genotype affects endogenous cluster levels. We asked whether other factors, i.e., in the cellular microenvironment, affect endogenous cluster levels and composition in hematopoietic cells. TK6 and WI-L2-NS cells were grown in standard medium (RPMI 1640) alone or supplemented with folate and/or selenium; oxidized base cluster levels were highest in RPMI 1640 and reduced in selenium-supplemented medium. Abasic clusters were low under all conditions. In primary hematopoietic stem and progenitor cells from four non-tobacco-using donors, cluster levels were low. However, in cells from tobacco users, we observed high oxidized base clusters and also abasic clusters, previously observed only in irradiated cells. Protein levels and activity of the abasic endonuclease Ape1 were similar in the tobacco users and nonusers. These data suggest that in highly damaging environments, even normal DNA repair capacity can be overwhelmed, leaving highly repair-resistant clustered damages.     

Sorting the consequences of ionizing radiation: processing of 8-oxoguanine/abasic site lesions

Clustered DNA damage is a hallmark of ionizing radiation. These complex lesions, composed of any combination of oxidized bases, abasic sites, or strand breaks within one helical turn, create a tremendous challenge for the base excision repair system, which must process the damage without generating cytotoxic double strand breaks (DSB). Clustered lesions affect the DNA incision activity of DNA glycosylases and AP endonucleases. Different levels of enzyme inhibition are dependent on lesion identity, orientation and separation. Very little is known about the simultaneous action of both classes of enzymes, which may lead to the creation of DSB. We have developed a novel substrate system of double-labeled hairpin duplexes, which allows the simultaneous determination of enzyme incision and formation of DBS. We use this system to study the processing of four clustered 8-oxoguanine/abasic site lesions by purified mouse Ogg1, human Ape1 and mouse embryonic stem cell nuclear extracts. Ape1 activity is least affected by the presence of a nearby oxidized base. In contrast, an abasic site inhibits the glycosylase and lyase activities of Ogg1 in an orientation-dependent manner. The combined action of both enzymes leads to the preferential formation of DSB with 5'-overhang ends. Processing of clusters by nuclear extracts displayed similar patter of enzyme inhibition and the same preference for avoiding double strand breaks with 3'-overhang ends.     

Processing of abasic DNA clusters in hApeI-silenced primary fibroblasts exposed to low doses of X-irradiation

Clustered damage in DNA includes two or more closely spaced oxidized bases, strand breaks or abasic sites that are induced by high- or low-linear-energy-transfer (LET) radiation, and these have been found to be repair-resistant and potentially mutagenic. In the present study we found that abasic clustered damages are also induced in primary human fibroblast cells by low-LET X-rays even at very low doses. In response to the induction of the abasic sites, primary fibroblasts irradiated by low doses of X-rays in the range 10–100 cGy showed dose-dependent up-regulation of the DNA repair enzyme, ApeI. We found that the abasic clusters in primary fibroblasts were more lethal to cells when hApeI enzyme expression was down-regulated by transfecting primary fibroblasts with hApeI siRNA as determined by clonogenic survival assay. Endonuclease activity of hApeI was found to be directly proportional to hApeI gene-silencing efficiency. The DNA repair profile showed that processing of abasic clusters was delayed in hApeI-siRNA-silenced fibroblasts, which challenges the survival of the cells even at very low doses of X-rays. Thus, the present study is the first to attempt to understand the induction of cluster DNA damage at very low doses of low-LET radiation in primary human fibroblasts and their processing by DNA repair enzyme ApeI and their relation with the survival of the cells.     

New insights into the structure of abasic DNA from molecular dynamics simulations

Abasic (AP) sites constitute a common form of DNA damage, arising from the spontaneous or enzymatic breakage of the N-glycosyl bond and the loss of a nucleotide base. To examine the effects of such damage on DNA structure, especially in the vicinity of the abasic sugar, four 1.5 ns molecular dynamics simulations of double-helical DNA dodecamers with and without a single abasic (tetrahydrofuran, X) lesion in a 5′-d(CXT) context have been performed and analyzed. The results indicate that the abasic site does not maintain a hole or gap in the DNA, but instead perturbs the canonical structure and induces additional flexibility close to the abasic site. In the apurinic simulations (i.e., when a pyrimidine is opposite the AP site), the abasic sugar flipped in and out of the minor groove, and the gap was water filled, except during the occurrence of a novel non-Watson–Crick C-T base pair across the abasic site. The apyrimidinic gap was not penetrated by water until the abasic sugar flipped out and remained extrahelical. Both AP helices showed kinks of 20–30° at the abasic site. The Watson–Crick hydrogen bonds are more transient throughout the DNA double helices containing an abasic site. The abasic sugar displayed an unusually broad range of sugar puckers centered around the northern pucker. The increased motion of the bases and backbone near the abasic site appear to correlate with sequence-dependent helical stability. The data indicate that abasic DNA contorts more easily and in specific ways relative to unmodified DNA, an aspect likely to be important in abasic site recognition and hydrolysis.     

High efficiency detection of bi-stranded abasic clusters in gamma-irradiated DNA by putrescine

Bi-stranded abasic clusters, an abasic (AP) site on one DNA strand and another nearby AP site or strand break on the other, have been quantified using Nfo protein from Escherichia coli to produce a double-strand break at cluster sites. Since recent data suggest that Nfo protein cleaves inefficiently at some clusters, we tested whether polyamines, which also cut at AP sites, would cleave abasic clusters at higher efficiency. The data show that Nfo protein cleaves poorly at clusters containing immediately opposed AP sites and those separated by 1 or 3 bp. Putrescine (PUTR) cleaved more efficiently than spermidine or spermine, and did not cleave undamaged DNA. It cleaved abasic clusters in oligonucleotide duplexes more effectively than Nfo protein, including immediately opposed or closely spaced clusters. PUTR cleaved more efficiently than Nfo protein by a factor of ~1.7 or ~2 for DNA that had been γ-irradiated in moderate or non-radioquenching conditions, respectively. This suggests that the DNA environment during irradiation affects the spectrum of cluster configurations. Further comparison of PUTR and Nfo protein cleavage may provide useful information on abasic cluster levels and configurations induced by ionizing radiation.     

Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.     

Mechanism of mutation on DNA templates containing synthetic abasic sites: study with a double strand vector.

Mutagenesis at abasic sites was investigated in E.coli and simian kidney (COS) cells using a duplex shuttle vector containing synthetic analogs of deoxyribose on the phosphodiester backbone. Lesions were positioned on opposite strands of the vector. When the tetrahydrofuranyl analog was used as the abasic site, AT or TA pairs (65-80%) were introduced at the site of the bistrand lesion. Mutagenesis occurred in the absence of SOS induction. Single base deletions (> 80%) dominated the mutational spectra for propanyl and ethanyl analogs of abasic sites lacking a ring structure. For all abasic site analogs, a small proportion of G/C and C/G pairs (6-10%) were observed. dAMP was incorporated predominantly opposite tetrahydrofuranyl sites positioned in the single strand region of a gapped duplex vector. We conclude from these studies that abasic sites positioned in a bistrand configuration are highly mutagenic in E.coli and COS cells. Repair DNA synthesis may be involved in this process.     

Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites.

The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via beta-elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism.     

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.     

Nucleotide insertion kinetics opposite abasic lesions in DNA

A gel assay is introduced to measure DNA polymerase insertion kinetics at single sites along a DNA template strand. The assay is used to analyze the kinetics of inserting deoxynucleotides opposite a synthetic abasic (apurinic/apyrimidinic) lesions using Drosophila DNA polymerase alpha. The location of the abasic lesion next to different nearest-neighbor bases allows the effects of base stacking on the specificity of insertion to be evaluated. The specificity of nucleotide insertion, Vmax/Km, is 6-11 times greater for A over G and about 20-50 times greater for A over C and T. The insertion specificity at the abasic lesion appears to depend more on differences in Vmax than Km. Apparent Michaelis constants for inserting A and G deoxynucleotides are similar to within about a factor of 2. The insertion of A or G occurs most efficiently at the abasic lesion when T is the 5'-nearest neighbor on the primer strand and least efficiently when G is the 5'-nearest neighbor. The presence of different base stacking partners adjacent to the site of insertion has up to a 4-fold effect on specificity.     

Correlation of bistranded clustered abasic DNA lesion processing with structural and dynamic DNA helix distortion

Clustered apurinic/apyrimidinic (AP; abasic) DNA lesions produced by ionizing radiation are by far more cytotoxic than isolated AP lesion entities. The structure and dynamics of a series of seven 23-bp oligonucleotides featuring simple bistranded clustered damage sites, comprising of two AP sites, zero, one, three or five bases 3′ or 5′ apart from each other, were investigated through 400 ns explicit solvent molecular dynamics simulations. They provide representative structures of synthetically engineered multiply damage sites-containing oligonucleotides whose repair was investigated experimentally (Nucl. Acids Res. 2004, 32:5609-5620; Nucl. Acids Res. 2002, 30: 2800–2808). The inspection of extrahelical positioning of the AP sites, bulge and non Watson–Crick hydrogen bonding corroborates the experimental measurements of repair efficiencies by bacterial or human AP endonucleases Nfo and APE1, respectively. This study provides unprecedented knowledge into the structure and dynamics of clustered abasic DNA lesions, notably rationalizing the non-symmetry with respect to 3′ to 5′ position. In addition, it provides strong mechanistic insights and basis for future studies on the effects of clustered DNA damage on the recognition and processing of these lesions by bacterial or human DNA repair enzymes specialized in the processing of such lesions.     

Defect of Dpb2p, a noncatalytic subunit of DNA polymerase ɛ, promotes error prone replication of undamaged chromosomal DNA in Saccharomyces cerevisiae

The biological consequences of clusters containing a single strand break and base lesion(s) remain largely unknown. In the present study we determined the mutagenicities of two- and three-lesion clustered damage sites containing a 1-nucleotide gap (GAP) and 8-oxo-7,8-dihydroguanine(s) (8-oxoG(s)) in Escherichia coli. The mutation frequencies (MFs) of bi-stranded two-lesion clusters (GAP/8-oxoG), especially in mutY-deficient strains, were high and were similar to those for bi-stranded clusters with 8-oxoG and base lesions/AP sites, suggesting that the GAP is processed with an efficiency similar to the efficiency of processing a base lesion or an AP site within a cluster. The MFs of tandem two-lesion clusters comprised of a GAP and an 8-oxoG on the same strand were comparable to or less than the MF of a single 8-oxoG. The mutagenic potential of three-lesion clusters, which were comprised of a tandem lesion (a GAP and an 8-oxoG) and an opposing single 8-oxoG, was higher than that of a single 8-oxoG, but was no more than that of a bi-stranded 8-oxoGs. We suggest that incorporation of a nucleotide opposite 8-oxoG is less mutagenic when a GAP is present in a cluster than when a GAP is absent. Our observations indicate that the repair of a GAP is retarded by an opposing 8-oxoG, but not by a tandem 8-oxoG, and that the extent of GAP repair determines the biological consequences.     

Integrity of duplex structures without hydrogen bonding: DNA with pyrene paired at abasic sites

DNA polymerases specifically insert the hydrophobic pyrene deoxynucleotide (P) opposite tetrahydrofuran (F), an stable abasic site analog, and DNA duplexes containing this non-hydrogen-bonded pair possess a high degree of thermodynamic stability. These observations support the hypothesis that steric complementarity and stacking interactions may be sufficient for maintaining stability of DNA structure and specificity of DNA replication, even in the absence of hydrogen bonds across the base pair. Here we report the NMR characterization and structure determination of two DNA molecules containing pyrene residues. The first is a 13mer duplex with a pyrene·tetrahydrofuran pair (P·F pair) at the ninth position and the second mimics a replication intermediate right after incorporation of a pyrene nucleoside opposite an abasic site. Our data indicate that both molecules adopt right-handed helical conformations with Watson– Crick alignments for all canonical base pairs. The pyrene ring stays inside the helix close to its baseless partner in both molecules. The single-stranded region of the replication intermediate folds back over the opposing strand, sheltering the hydrophobic pyrene moiety from water exposure. The results support the idea that the stability and replication of a P·F pair is due to its ability to mimic Watson–Crick structure.     

Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease?1

Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo–helical ‘flexicate’ complexes, bimetallic triple-stranded ferro-helicates [Fe₂(NN-NN)₃]⁴⁺ incorporating the common NN–NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo–helical complexes for cancer chemotherapy.     

Human AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end-joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3′ exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K^27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme’s activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.