Deoxyribose Phosphate Lyase Activity Research Articles

Mitochondrial transcription factor A (TFAM) has 5′-deoxyribose phosphate lyase activity in vitro

Mitochondrial DNA (mtDNA) plays a key role in mitochondrial and cellular functions. mtDNA is maintained by active DNA turnover and base excision repair (BER). In BER, one of the toxic repair intermediates is 5′-deoxyribose phosphate (5′dRp). Human mitochondrial DNA polymerase γ has weak dRp lyase activities, and another known dRp lyase in the nucleus, human DNA polymerase β, can also localize to mitochondria in certain cell and tissue types. Nonetheless, whether additional proteins have the ability to remove 5'dRp in mitochondria remains unknown. Our prior work on the AP lyase activity of mitochondrial transcription factor A (TFAM) has prompted us to examine its ability to remove 5′dRp residues in vitro. TFAM is the primary DNA-packaging factor in human mitochondria and interacts with mitochondrial DNA extensively. Our data demonstrate that TFAM has the dRp lyase activity with different DNA substrates. Under single-turnover conditions, TFAM removes 5′dRp residues at a rate comparable to that of DNA polymerase (pol) β, albeit slower than that of pol λ. Among the three proteins examined, pol λ shows the highest single-turnover rates in dRp lyase reactions. The catalytic effect of TFAM is facilitated by lysine residues of TFAM via Schiff base chemistry, as evidenced by the observation of dRp-lysine adducts in mass spectrometry experiments. The catalytic effect of TFAM observed here is analogous to the AP lyase activity of TFAM reported previously. Together, these results suggest a potential role of TFAM in preventing the accumulation of toxic DNA repair intermediates.

5'-Deoxyribose Phosphate Lyase Activity of Apurinic/Apyrimidinic Endonuclease 1

5'-Deoxyribose Phosphate Lyase Activity of Apurinic/Apyrimidinic Endonuclease 1

5'-Deoxyribose Phosphate Lyase Activity of Apurinic/Apyrimidinic Endonuclease 1

One of the most common DNA lesions is the appearance of apurinic/apyrimidinic (AP-) sites. The main repair pathway for AP sites is initiated by apurinic/apyrimidinic endonuclease 1 (APE1). Upon hydrolysis of the phosphodiester bond by this enzyme, a one nucleotide gap flanked by 3'-hydroxyl and 5'-deoxyribose phosphate groups on the 5'-side of the AP site is formed. After hydrolysis of the AP site, APE1 remains associated with the product for some time. In the present work, the ability of APE1 to form a product of covalent attachment of APE1 to DNA containing a gap with a 5'-deoxyribose phosphate residue was demonstrated. In addition, it was found that while in a complex with the product of hydrolysis of the AP site, APE1 exhibits 5'-deoxyribose phosphate lyase activity, cleaving off the 5'-deoxyribose phosphate residue. The presence of lyase activity in APE1 may be important for the repair of AP sites if there is a deficiency of, or mutations in DNA polymerase β, the main enzyme that removes the 5'-deoxyribose phosphate group.

Three Human Pol ι Variants with Impaired Polymerase Activity Fail to Rescue H2O2 Sensitivity in POLI-Deficient Cells.

Human Y-family DNA polymerase (pol) ι is involved in translesion DNA synthesis (TLS) and base excision repair (BER) of oxidative DNA damage. Genetic variations may alter the function of pol ι and affect cellular susceptibility to oxidative genotoxic agents, but their effects remain unclear. We investigated the impacts of 10 human missense germline variations on pol ι function by biochemical and cell-based assays. Both polymerase and deoxyribose phosphate (dRP) lyase activities were determined utilizing recombinant pol ι (residues 1-445) proteins. The K209Q, K228I, and Q386R variants showed 4- to 53-fold decreases in specificity constants (kcat/Km) for dCTP insertion opposite G and 8-oxo-7,8-dihydroguanine compared to the wild-type. The R126C and K345E variants showed wild-type-like polymerase activity, although these two variants (as well as the R209Q, K228I, and Q386R variants) showed greater than 6-fold decreases in dRP lyase activity compared to the wild-type. A CRISPR/Cas9-mediated POLI knockout conferred higher sensitivity to H2O2 in human embryonic kidney (HEK293) cells. Exogenous expression of the full-length wild-type, R126C, and K345E variants fully rescued the H2O2 sensitivity in POLI-deficient cells, while full-length R209Q, K228I, and Q386R variants did not rescue the sensitivity. Our results indicate that the R126C and K345E variants (having wild-type-like polymerase activity, albeit impaired in dRP lyase activity) could fully rescue the H2O2 sensitivity in POLI-deficient cells, while the R209Q, K228I, and Q386R variants, all impaired in polymerase and dRP lyase activity, failed to rescue the sensitivity, indicating the relative importance of TLS-related polymerase function of pol ι rather than its BER-related dRP lyase function in protection from oxidative stress. The possibility exists that the hypoactive pol ι variants increase the individual susceptibility to oxidative genotoxic agents.

Molecular dynamics simulations suggest changes in electrostatic interactions as a potential mechanism through which serine phosphorylation inhibits DNA polymerase β activity

DNA polymerase β is a 39 kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31 kDa domain responsible for the polymerase activity and an 8 kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics simulations. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S55/44 and S55 phosphorylation were less dramatic and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is the major contributor to structural fluctuations that lead to loss of enzymatic activity.

The deoxyribose phosphate lyase of DNA polymerase β suppresses a processive DNA synthesis to prevent trinucleotide repeat instability.

Trinucleotide repeat (TNR) instability is associated with over 42 neurodegenerative diseases and cancer, for which the molecular mechanisms remain to be elucidated. We have shown that the DNA base excision repair (BER) pathway and its central component, DNA polymerase β (pol β), in particular, its polymerase activity plays an active role in regulating somatic TNR instability. Herein, we revealed a unique role of the pol β dRP lyase in preventing somatic TNR instability. We found that deficiency of pol β deoxyribose phosphate (dRP) lyase activity locked the pol β dRP lyase domain to a dRP group, and this ‘tethered’ pol β to its template forcing the polymerase to perform a processive DNA synthesis. This subsequently promoted DNA strand slippage allowing pol β to skip over a template loop and causing TNR deletion. We showed that the effects were eliminated by complementation of the dRP lyase deficiency with wild-type pol β protein. The results indicate that pol β dRP lyase activity restrained the pol β-dRP interaction to suppress a pol β processive DNA synthesis, thereby preventing TNR deletion. This further implicates a potential of pol β dRP lyase inhibition as a novel treatment of TNR-expansion diseases.

Publisher’s Note

DNA polymerase β is a 39kDa enzyme that is a major component of Base Excision Repair in human cells. The enzyme comprises two major domains, a 31kDa domain responsible for the polymerase activity and an 8kDa domain, which bind ssDNA and has a deoxyribose phosphate (dRP) lyase activity. DNA polymerase β was shown to be phosphorylated in vitro with protein kinase C (PKC) at serines 44 and 55 (S44 and S55), resulting in loss of its polymerase enzymic activity, but not its ability to bind ssDNA. In this study, we investigate the potential phosphorylation-induced structural changes for DNA polymerase β using molecular dynamics. The simulations show drastic conformational changes of the polymerase structure as a result of S44 phosphorylation. Phosphorylation-induced conformational changes transform the closed (active) enzyme structure into an open one. Further analysis of the results points to a key hydrogen bond and newly formed salt bridges as potential drivers of these structural fluctuations. The changes observed with S44/55 and S55 phosphorylation were less dramatic than S44 and the integrity of the H-bond was not compromised. Thus the phosphorylation of S44 is likely the major contributor to structural fluctuations that lead to loss of enzymatic activity.

Rev1 is a base excision repair enzyme with 5'-deoxyribose phosphate lyase activity.

Rev1 is a member of the Y-family of DNA polymerases and is known for its deoxycytidyl transferase activity that incorporates dCMP into DNA and its ability to function as a scaffold factor for other Y-family polymerases in translesion bypass events. Rev1 also is involved in mutagenic processes during somatic hypermutation of immunoglobulin genes. In light of the mutation pattern consistent with dCMP insertion observed earlier in mouse fibroblast cells treated with a base excision repair-inducing agent, we questioned whether Rev1 could also be involved in base excision repair (BER). Here, we uncovered a weak 5′-deoxyribose phosphate (5′-dRP) lyase activity in mouse Rev1 and demonstrated the enzyme can mediate BER in vitro. The full-length Rev1 protein and its catalytic core domain are similar in their ability to support BER in vitro. The dRP lyase activity in both of these proteins was confirmed by NaBH4 reduction of the Schiff base intermediate and kinetics studies. Limited proteolysis, mass spectrometry and deletion analysis localized the dRP lyase active site to the C-terminal segment of Rev1's catalytic core domain. These results suggest that Rev1 could serve as a backup polymerase in BER and could potentially contribute to AID-initiated antibody diversification through this activity.

Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIalpha-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions -1 and -2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.

Potentiation of Temozolomide Cytotoxicity by Inhibition of DNA Polymerase β Is Accentuated by BRCA2 Mutation

Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

BackgroundDNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB).Principal FindingsHere, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ.ConclusionsThe expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.

DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase beta. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5'-deoxyribose phosphate (5'-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5'-dRP lyase and a truncated polymerase domain was comparatively less sensitive to gamma-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIalpha-XRCC1 complex. Of these, ligase IIIalpha-XRCC1 is the most efficient. Moreover, ligase IIIalpha-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.

Reconstitution of uracil DNA glycosylase initiated base excision repair in herpes simplex virus‐1

Herpes simplex virus‐1 is self‐sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV‐1 DNA polymerase (UL30) exhibits AP and 5′‐deoxyribose phosphate lyase activities that are integral to base excision repair (BER). We have mapped the lyase activity to the C‐terminal polymerase domain of UL30. This domain lacks polymerase activity but binds DNA. Candidate lysine residues in this fragment will be mutagenized to identify the lyase active site. We have also reconstituted a system with purified HSV‐1 and human proteins that performs all the steps of uracil DNA glycosylase initiated BER. In this system, nucleotide incorporation is dependent on the HSV‐1 uracil DNA glycosylase, AP endonuclease and UL30. Completion of BER is mediated by DNA ligase IIIα/XRCC1, which also confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV‐1 DNA polymerase processivity factor and prevents BER from occurring with heterologous DNA polymerases. These findings demonstrate that the HSV‐1 proteins in combination with cellular factors are capable of performing BER and have implications on viral genome maintenance during lytic replication and reactivation from latency.This work was supported by grant GM 062643 from the NIH.

Human DNA polymerase possesses 5'-dRP lyase activity and functions in single-nucleotide base excision repair in vitro

DNA polymerase θ (Pol θ) is a low-fidelity DNA polymerase that belongs to the family A polymerases and has been proposed to play a role in somatic hypermutation. Pol θ has the ability to conduct translesion DNA synthesis opposite an AP site or thymine glycol, and it was recently proposed to be involved in base excision repair (BER) of DNA damage. Here, we show that Pol θ has intrinsic 5′-deoxyribose phosphate (5′-dRP) lyase activity that is involved in single-nucleotide base excision DNA repair (SN-BER). Full-length human Pol θ is a ∼300-kDa polypeptide, but we show here that the 98-kDa C-terminal region of Pol θ possesses both DNA polymerase activity and dRP lyase activity and is sufficient to carry out base excision repair in vitro. The 5′-dRP lyase activity is independent of the polymerase activity, in that a polymerase inactive mutant retained full 5′-dRP lyase activity. Domain mapping of the 98-kDa enzyme by limited proteolysis and NaBH4 cross-linking with a BER intermediate revealed that the dRP lyase active site resides in a 24-kDa domain of Pol θ. These results are consistent with a role of Pol θ in BER.

Human DNA polymerase iota protects cells against oxidative stress

Human DNA polymerase iota (poliota) is a unique member of the Y-family of specialised polymerases that displays a 5'deoxyribose phosphate (dRP) lyase activity. Although poliota is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biological importance of poliota in human cells, we generated fibroblasts stably downregulating poliota (MRC5-pol iota(KD)) and examined their response to several types of DNA-damaging agents. We show that cell lines downregulating poliota exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H(2)O(2)) or menadione but not to ethylmethane sulphonate (EMS), UVC or UVA. Interestingly, extracts from cells downregulating poliota show reduced base excision repair (BER) activity. In addition, poliota binds to chromatin after treatment of cells with H(2)O(2) and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged poliota accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human poliota in protecting cells from oxidative damage.

Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance

Mammalian DNA polymerase β is a nuclear enzyme involved in the base excision and single-stranded DNA break repair pathways. In trypanosomatids, this protein does not have a defined cellular localization, and its function is poorly understood. We characterized two Trypanosoma cruzi proteins homologous to mammalian DNA polymeraseβ, TcPolβ and TcPolβPAK, and showed that both enzymes localize to the parasite kinetoplast. In vitro assays with purified proteins showed that they have DNA polymerization and deoxyribose phosphate lyase activities. Optimal conditions for polymerization were different for each protein with respect to dNTP concentration and temperature, and TcPolβPAK, in comparison to TcPolβ, conducted DNA synthesis over a much broader pH range. TcPolβ was unable to carry out mismatch extension or DNA synthesis across 8-oxodG lesions, and was able to discriminate between dNTP and ddNTP. These specific abilities of TcPolβ were not observed for TcPolβPAK or other X family members, and are not due to a phenylalanine residue at position 395 in the C-terminal region of TcPolβ, as assessed by a site-directed mutagenesis experiment reversing this residue to a well conserved tyrosine. Our data suggest that both polymerases from T. cruzi could cooperate to maintain mitochondrial DNA integrity through their multiple roles in base excision repair, gap filling and translesion synthesis.

The replicative DNA polymerase of herpes simplex virus 1 exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities

Base excision repair (BER) is essential for maintaining genome stability both to counter the accumulation of unusual bases and to protect from base loss in the DNA. Herpes simplex virus 1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery, including enzymes involved in nucleotide metabolism. We report on a replicative family B and a herpesvirus-encoded DNA Pol that possesses DNA lyase activity. We have discovered that the catalytic subunit of the HSV-1 DNA polymerase (Pol) (UL30) exhibits apurinic/apyrimidinic (AP) and 5'-deoxyribose phosphate (dRP) lyase activities. These activities are integral to BER and lead to DNA cleavage on the 3' side of abasic sites and 5'-dRP residues that remain after cleavage by 5'-AP endonuclease. The UL30-catalyzed reaction occurs independently of divalent cation and proceeds via a Schiff base intermediate, indicating that it occurs via a lyase mechanism. Partial proteolysis of the Schiff base shows that the DNA lyase activity resides in the Pol domain of UL30. These observations together with the presence of a virus-encoded uracil DNA glycosylase indicates that HSV-1 has the capacity to perform critical steps in BER. These findings have implications on the role of BER in viral genome maintenance during lytic replication and reactivation from latency.

XRCC1 and DNA polymerase β in cellular protection against cytotoxic DNA single-strand breaks

Single-strand breaks (SSBs) can occur in cells either directly, or indirectly following initiation of base excision repair (BER). SSBs generally have blocked termini lacking the conventional 5'-phosphate and 3'-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1(-/-) mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase beta (pol beta) is specific to this pathway, whereas pol beta is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS-treated XRCC1(-/-), and to a lesser extent in pol beta(-/-) cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and pol beta(-/-) cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1(-/-) cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly(ADP-ribosyl)ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC1 to sites of DNA damage.