Base Excision Repair Intermediates Research Articles

Structural insight into the DNA polymerase β deoxyribose phosphate lyase mechanism

A large number of biochemical and genetic studies have demonstrated the involvement of DNA polymerase β (Pol β) in mammalian base excision repair (BER). Pol β participates in BER sub-pathways by contributing gap filling DNA synthesis and lyase removal of the 5′-deoxyribose phosphate (dRP) group from the cleaved abasic site. To better understand the mechanism of the dRP lyase reaction at an atomic level, we determined a crystal structure of Pol β complexed with 5′-phosphorylated abasic sugar analogs in nicked DNA. This DNA ligand represents a potential BER intermediate. The crystal structure reveals that the dRP group is bound in a non-catalytic binding site. The catalytic nucleophile in the dRP lyase reaction, Lys72, and all other potential secondary nucleophiles, are too far away to participate in nucleophilic attack on the C1′ of the sugar. An approximate model of the dRP group in the expected catalytic binding site suggests that a rotation of 120° about the dRP 3′-phosphate is required to position the ɛ-amino Lys72 close to the dRP C1′. This model also suggests that several other side chains are in position to facilitate the β-elimination reaction. From results of mutational analysis of key residues in the dRP lyase active site, it appears that the substrate dRP can be stabilized in the observed non-catalytic binding conformation, hindering dRP lyase activity.

Base excision repair intermediates are mutagenic in mammalian cells

Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5′-deoxyribose-5-phosphate (5′dRp) and 3′-[2,3-didehydro-2,3-dideoxy-ribose] (3′ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the ‘A-rule’ and is also consistent with the use of the 5′ neighbouring base to instruct nucleotide incorporation (5′-rule). Preferential adenine insertion was also observed after in vivo replication of 5′dRp or 3′ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives ‘faceless’ BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations.

The Role of Base Excision Repair in the Sensitivity and Resistance to Temozolomide-Mediated Cell Death

DNA-alkylating agents have a central role in the curative therapy of many human tumors; yet, resistance to these agents limits their effectiveness. The efficacy of the alkylating agent temozolomide has been attributed to the induction of O6-MeG, a DNA lesion repaired by the protein O6-methylguanine-DNA methyltransferase (MGMT). Resistance to temozolomide has been ascribed to elevated levels of MGMT and/or reduced mismatch repair. However, >80% of the DNA lesions induced by temozolomide are N-methylated bases that are recognized by DNA glycosylases and not by MGMT, and so resistance to temozolomide may also be due, in part, to robust base excision repair (BER). We used isogenic cells deficient in the BER enzymes DNA polymerase-beta (pol-beta) and alkyladenine DNA glycosylase (Aag) to determine the role of BER in the cytotoxic effect of temozolomide. Pol-beta-deficient cells were significantly more susceptible to killing by temozolomide than wild-type or Aag-deficient cells, a hypersensitivity likely caused by accumulation of BER intermediates. RNA interference-mediated pol-beta suppression was sufficient to increase temozolomide efficacy, whereas a deficiency in pol-iota or pol-lambda did not increase temozolomide-mediated cytotoxicity. Overexpression of Aag (the initiating BER enzyme) triggered a further increase in temozolomide-induced cytotoxicity. Enhanced Aag expression, coupled with pol-beta knockdown, increased temozolomide efficacy up to 4-fold. Furthermore, loss of pol-beta coupled with temozolomide treatment triggered the phosphorylation of H2AX, indicating the activation of the DNA damage response pathway as a result of unrepaired lesions. Thus, the BER pathway is a major contributor to cellular resistance to temozolomide and its efficacy depends on specific BER gene expression and activity.

Comparison of functional properties of mammalian DNA polymerase λ and DNA polymerase β in reactions of DNA synthesis related to DNA repair

DNA polymerase λ (Pol λ) is a novel enzyme of the family X of DNA polymerases. Pol λ has some properties in common with DNA polymerase β (Pol β). The substrate properties of Pol λ were compared to Pol β using DNAs mimicking short-patch (SP) and long-patch (LP) base excision repair (BER) intermediates as well as recessed template primers. In the present work, the influence of several BER proteins such as flap-endonuclease-1 (FEN1), PCNA, and apurinic/apyrimidinic endonuclease-1 (APE1) on the activity of Pol λ was investigated. Pol λ is unable to catalyze strand displacement synthesis using nicked DNA, although this enzyme efficiently incorporates a dNMP into a one-nucleotide gap. FEN1 and PCNA stimulate the strand displacement activity of Pol λ. FEN1 processes nicked DNA, thus removing a barrier to Pol λ DNA synthesis. It results in a one-nucleotide gapped DNA molecule that is a favorite substrate of Pol λ. Photocrosslinking and functional assay show that Pol λ is less efficient than Pol β in binding to nicked DNA. APE1 has no influence on the strand displacement activity of Pol λ though it stimulates strand displacement synthesis catalyzed with Pol β. It is suggested that Pol λ plays a role in the SP BER rather than contributes to the LP BER pathway.

Oxidative damage in telomeric DNA disrupts recognition by TRF1 and TRF2

The ends of linear chromosomes are capped by protein–DNA complexes termed telomeres. Telomere repeat binding factors 1 and 2 (TRF1 and TRF2) bind specifically to duplex telomeric DNA and are critical components of functional telomeres. Consequences of telomere dysfunction include genomic instability, cellular apoptosis or senescence and organismal aging. Mild oxidative stress induces increased erosion and loss of telomeric DNA in human fibroblasts. We performed binding assays to determine whether oxidative DNA damage in telomeric DNA alters the binding activity of TRF1 and TRF2 proteins. Here, we report that a single 8-oxo-guanine lesion in a defined telomeric substrate reduced the percentage of bound TRF1 and TRF2 proteins by at least 50%, compared with undamaged telomeric DNA. More dramatic effects on TRF1 and TRF2 binding were observed with multiple 8-oxo-guanine lesions in the tandem telomeric repeats. Binding was likewise disrupted when certain intermediates of base excision repair were present within the telomeric tract, namely abasic sites or single nucleotide gaps. These studies indicate that oxidative DNA damage may exert deleterious effects on telomeres by disrupting the association of telomere-maintenance proteins TRF1 and TRF2.

Regulation of WRN Helicase Activity in Human Base Excision Repair

Werner syndrome patients are deficient in the Werner protein (WRN), which is a multifunctional nuclear protein possessing 3'-5' exonuclease and ATP-dependent helicase activities. Studies of Werner syndrome cells and biochemical studies of WRN suggest that WRN plays a role in several DNA metabolic pathways. WRN interacts with DNA polymerase beta (pol beta) and stimulates pol beta strand displacement synthesis on a base excision repair (BER) intermediate in a helicase-dependent manner. In this report, we examined the effect of the major human apurinic/apyrimidinic endonuclease (APE1) and of pol beta on WRN helicase activity. The results show that WRN alone is able to unwind several single strand break BER intermediates. However, APE1 inhibits WRN helicase activity on these intermediates. This inhibition is likely due to the binding of APE1 to nicked apurinic/apyrimidinic sites, suggesting that APE1 prevents the promiscuous unwinding of BER intermediates. This inhibitory effect was relieved by the presence of pol beta. A model involving the pol beta-mediated hand-off of WRN protein is proposed based on these results.

Transient adenoviral N-methylpurine DNA glycosylase overexpression imparts chemotherapeutic sensitivity to human breast cancer cells

Abstract In an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change, we have used adenoviral overexpression of N-methylpurine DNA glycosylase (MPG or ANPG/AAG) in breast cancer cells to study its ability to imbalance base excision repair (BER) and sensitize cancer cells to alkylating agents. Our results show that MPG-overexpressing cells are significantly more sensitive to the alkylating agents methyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, methylnitrosourea, dimethyl sulfate, and the clinical chemotherapeutic temozolomide. Sensitivity is further increased through coadministration of the BER inhibitor methoxyamine, which covalently binds abasic or apurinic/apyrimidinic (AP) sites and makes them refractory to subsequent repair. Methoxyamine reduction of cell survival is significantly greater in cells overexpressing MPG than in control cells, suggesting a heightened production of AP sites that, if made persistent, results in increased cellular toxicity. We further explored the mechanism of MPG-induced sensitivity and found that sensitivity was associated with a significant increase in the number of AP sites and/or single-strand breaks in overexpressing cells, confirming a MPG-driven accumulation of toxic BER intermediates. These data establish transient MPG overexpression as a potential therapeutic approach for increasing cellular sensitivity to alkylating agent chemotherapy.

Poly(ADP-ribose) polymerase-1 inhibits strand-displacement synthesis of DNA catalyzed by DNA polymerase beta.

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.

281. Modulating the Immune Response to Oncolytic Measles Viruses

Top of pageAbstract In an effort to improve the efficacy of cancer chemotherapy by intervening into the cellular responses to chemotherapeutic change, we have used adenoviral overexpression of N-methylpurine DNA glycosylase (MPG, ANPG or AAG) in breast cancer cells, to study its ability to imbalance base excision repair (BER) and sensitize cancer cells to alkylating agents. Our results demonstrate that MPG overexpressing cells are significantly more sensitive to the alkylating agents methyl methanesulfonate (MMS), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), methyl nitrosourea (MNU), dimethyl sulfate (DMS) and the clinical chemotherapeutic temozolomide (TMZ). Sensitivity is further increased through co-administration of the BER inhibitor methoxyamine (MX), which covalently binds abasic (AP) sites and makes them refractory to subsequent repair. MX reduction of cell survival is significantly greater in cells overexpressing MPG than in control cells, suggesting a heightened production of AP sites that if made persistent, results in increased cellular toxicity. We further explored the mechanism of MPG-induced sensitivity, and found that sensitivity was associated with a significant increase in the number of AP sites and/or single strand breaks in overexpressing cells, confirming an MPG-driven accumulation of toxic BER intermediates. This data establishes transient MPG overexpression as a potential therapeutic approach for increasing cellular sensitivity to alkylating agent chemotherapy.

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ß (ß-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3′- to 5′-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5′-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.

Base Excision Repair Intermediates Induce p53-independent Cytotoxic and Genotoxic Responses

DNA alkylation damage is primarily repaired by the base excision repair (BER) machinery in mammalian cells. In repair of the N-alkylated purine base lesion, for example, alkyl adenine DNA glycosylase (Aag) recognizes and removes the base, and DNA polymerase beta (beta-pol) contributes the gap tailoring and DNA synthesis steps. It is the loss of beta-pol-mediated 5'-deoxyribose phosphate removal that renders mouse fibroblasts alkylation-hypersensitive. Here we report that the hypersensitivity of beta-pol-deficient cells after methyl methanesulfonate-induced alkylation damage is wholly dependent upon glycosylase-mediated initiation of repair, indicating that alkylated base lesions themselves are tolerated in these cells and demonstrate that beta-pol protects against accumulation of toxic BER intermediates. Further, we find that these intermediates are initially tolerated in vivo by a second repair pathway, homologous recombination, inducing an increase in sister chromatid exchange events. If left unresolved, these BER intermediates trigger a rapid block in DNA synthesis and cytotoxicity. Surprisingly, both the cytotoxic and genotoxic signals are independent of both the p53 response and mismatch DNA repair pathways, demonstrating that p53 is not required for a functional BER pathway, that the observed damage response is not part of the p53 response network, and that the BER intermediate-induced cytotoxic and genotoxic effects are distinct from the mechanism engaged in response to mismatch repair signaling. These studies demonstrate that, although base damage is repaired by the BER pathway, incomplete BER intermediates are shuttled into the homologous recombination pathway, suggesting possible coordination between BER and the recombination machinery.

Modulation of the 3′→5′-Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5′-incised Abasic DNA Product

The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta. To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta. However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta.

Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase ι by Controlled Proteolysis

Human DNA polymerase iota (pol iota) is a member of the Y-family of low fidelity lesion bypass DNA polymerases. In addition to a probable role in DNA lesion bypass, this enzyme has recently been shown to be required for somatic hypermutation in human B-cells. We found earlier that human pol iota has deoxyribose phosphate (dRP) lyase activity and unusual specificity for activity during DNA synthesis, suggesting involvement in specialized forms of base excision repair (BER). Here, mapping of the domain structure of human pol iota by controlled proteolysis revealed that the enzyme has a 48-kDa NH2-terminal domain and a protease resistant 40-kDa "core domain" spanning residues Met79 to approximately Met445. A covalently cross-linked pol iota-DNA complex, representing a trapped intermediate in the dRP lyase reaction, was subjected to controlled proteolysis. Cross-linking was mapped to the 40-kDa core domain, indicating that the dRP lyase active site is in this region. To further evaluate the BER capacity of the enzyme, the dRP lyase and DNA polymerase activities were characterized on DNA substrates representing BER intermediates, and we found that pol iota was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase beta null cell extract.

XRCC1 and DNA strand break repair

DNA single-strand breaks can arise indirectly, as normal intermediates of DNA base excision repair, or directly from damage to deoxyribose. Because single-strand breaks are induced by endogenous reactive molecules such as reactive oxygen species, these lesions pose a continuous threat to genetic integrity. XRCC1 protein plays a major role in facilitating the repair of single-strand breaks in mammalian cells, via an ability to interact with multiple enzymatic components of repair reactions. Here, the protein–protein interactions facilitated by XRCC1, and the repair processes in which these interactions operate, are reviewed. Models for the repair of single-strand breaks during base excision repair and at direct breaks are presented.

DNA Polymerase X of African Swine Fever Virus: Insertion Fidelity on Gapped DNA substrates and AP lyase Activity Support a Role in Base Excision Repair of Viral DNA

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3′–5′ exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5′-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5′-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10−4 to 10−5, and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5′-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.

A new binary system for photosensitized labeling of DNA polymerases in nuclear extract.

A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3;-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5;-phosphate) at the 5;-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase beta labeling.

Polymorphisms in the DNA repair gene XRCC1 and age-related disease

The recent hypothesis that common variants (single nucleotide polymorphisms or SNPs) in the population may contribute significantly to genetic risk for common diseases permits a conceptually straightforward approach to identifying age-related disease-causing mutations. Functional variants of DNA replication and repair genes might be expected to be highly significant to cancer and aging since replication must proceed with high fidelity in a cellular environment where an estimated 10 000 nucleotides are damaged daily. Single-strand breaks (SSB) are one of the results of DNA damage either by methylation, oxidation, reduction or fragmentation of bases by ionizing radiation, and arise in cells directly by disintegration of damaged sugars or indirectly as intermediates of base excision repair. Studies have demonstrated a role for XRCC1 both in vitro and in vivo during the repair of SSB. A number of SNPs have been identified for the XRCC1 gene, and several have been associated with age-related diseases, especially cancer. This report provides resequencing data confirming the existence of commonly occurring SNPs, including Arg194Trp and Arg399Gln, and briefly summarizes epidemiological and functional relevance to cancer and other age-related diseases. XRCC1 SNPs will be useful probes for investigating age-associated pathobiology in epidemiological and mechanistic studies.

Photoaffinity labeling of proteins in bovine testis nuclear extract

A binary system of photoaffinity reagents for selective affinity labeling of DNA polymerases has been developed. The photoreactive probe was formed in nuclear extract, using an end-labeled oligonucleotide containing a synthetic abasic site. This site was incised by apurinic/apyrimidinic endonuclease and then dNMPs carrying a photoreactive adduct were added to the 3 ′ hydroxyl using base-substituted arylazido derivatives of dUTP or dCTP. This results in the synthesis of photoreactive base excision repair (BER) intermediates. The photoreactive group was then activated, either directly (UV light exposure 320 nm) or in the presence of the sensitizer of dTTP analog containing a pyrene group (Pyr-dUTP) under UV light 365 nm. DNA polymerase β was the main target crosslinked by photoreactive BER intermediates in this nuclear extract. In contrast, several proteins were labeled under the conditions of direct activation of arylazido group.

Nitric oxide-induced homologous recombination in Escherichia coli is promoted by DNA glycosylases.

Nitric oxide (NO*) is involved in neurotransmission, inflammation, and many other biological processes. Exposure of cells to NO* leads to DNA damage, including formation of deaminated and oxidized bases. Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO* toxicity, which indicates that base excision repair (BER) intermediates are being generated. Here, we show that AP endonuclease-deficient cells can be protected from NO* toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase. These results suggest that Ung and Fpg remove nontoxic NO*-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases. Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination. The RecBCD complex is critical for repair of double-strand breaks via homologous recombination. When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced. We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO*-induced recombination. Taken together, a picture emerges in which the action of DNA glycosylases on NO*-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination. These studies shed light on the underlying mechanism of NO*-induced homologous recombination.

The S. cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination

DNA glycosylases, such as the Mag1 3-methyladenine (3MeA) DNA glycosylase, initiate the base excision repair (BER) pathway by removing damaged bases to create abasic apurinic/apyrimidinic (AP) sites that are subsequently repaired by downstream BER enzymes. Although unrepaired base damage may be mutagenic or recombinogenic, BER intermediates (e.g. AP sites and strand breaks) may also be problematic. To investigate the molecular basis for methylation-induced homologous recombination events in Saccharomyces cerevisiae, spontaneous and methylation-induced recombination were studied in strains with varied MAG1 expression levels. We show that cells lacking Mag1 have increased susceptibility to methylation-induced recombination, and that disruption of nucleotide excision repair (NER; rad4) in mag1 cells increases cellular susceptibility to these events. Furthermore, expression of Escherichia coli Tag 3MeA DNA glycosylase suppresses recombination events, providing strong evidence that unrepaired 3MeA lesions induce recombination. Disruption of REV3 (required for polymerase ζ (Pol ζ)) in mag1 rad4 cells causes increased susceptibility to methylation-induced toxicity and recombination, suggesting that Pol ζ can replicate past 3MeAs. However, at subtoxic levels of methylation damage, disruption of REV3 suppresses methylation-induced recombination, indicating that the effects of Pol ζ on recombination are highly dose-dependent. We also show that overproduction of Mag1 can increase the levels of spontaneous recombination, presumably due to increased levels of BER intermediates. However, additional APN1 endonuclease expression or disruption of REV3 does not affect MAG1-induced recombination, suggesting that downstream BER intermediates (e.g. single strand breaks) are responsible for MAG1-induced recombination, rather than uncleaved AP sites. Thus, too little Mag1 sensitizes cells to methylation-induced recombination, while too much Mag1 can put cells at risk of recombination induced by single strand breaks formed during BER.