DNA polymerase ß deficiency promotes the occurrence of esophageal precancerous lesions in mice.

Esophageal mucosa undergoes mild, moderate, severe dysplasia, and other precancerous lesions and eventually develops into carcinoma in situ, and understanding the developmental progress of esophageal precancerous lesions is beneficial to prevent them from developing into cancer. DNA polymerase ß (Polß), a crucial enzyme of the base excision repair system, plays an important role in repairing damaged DNA and maintaining genomic stability. Abnormal expression or deletion mutation of Polß is related to the occurrence of esophageal cancer, but the role of Polß deficiency in the esophageal precancerous lesions is still unclear. Here, esophageal mucosa Polß-knockout mice were used to explore the relationship of Polß deficiency with esophageal precancerous lesions. First, we found the degree and number of esophageal precancerous lesions in Polß-KO mice were more serious than those in Polß-Loxp mice after N-nitrosomethylbenzylamine (NMBA) treatment. Whole exome sequencing revealed that deletion of Polß increased the frequency of gene mutations. Gene expression prolife analysis showed that the expression of proteins correlated to cell proliferation and the cell cycle was elevated in Polß-KO mice. We also found that deletion of Polß promoted the proliferation and clone formation as well as accelerated cell cycle progression of human immortalized esophageal epithelial cell line SHEE treated with NMBA. Our findings indicate that Polß knockout promotes the occurrence of esophageal precancerous lesions.

Suppression of DNA Double-Strand Break Formation by DNA Polymerase ß in Active DNA Demethylation Is Required for Development of Hippocampal Pyramidal Neurons.

Genome stability is essential for brain development and function, as de novo mutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase ß (Polß) is involved in hippocampal pyramidal neuron differentiation via a TET-mediated active DNA demethylation during early postnatal stages using Nex-Cre/Polß fl/fl mice of either sex, in which forebrain postmitotic excitatory neurons lack Polß expression. Polß deficiency induced extensive DNA double-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polß maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory.SIGNIFICANCE STATEMENT Increasing evidence suggests that de novo mutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polß, a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polß deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break formation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aberrant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development.

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

A switch between DNA polymerases δ and λ promotes error-free bypass of 8-oxo-G lesions.

7,8-Dihydro-8-oxoguanine (8-oxo-G) is a highly abundant and mutagenic lesion. Replicative DNA polymerases (pols) are slowed down at 8-oxo-G and insert both correct cytosine (C) and incorrect adenine (A) opposite 8-oxo-G, but they preferentially extend A:8-oxo-G mispairs. Nevertheless, 8-oxo-G bypass is fairly accurate in vivo. Thus, the question how correct bypass of 8-oxo-G lesions is accomplished despite the poor extension of C:8-oxo-G base pairs by replicative pols remains unanswered. Here we show that replicative pol δ pauses in front of 8-oxo-G and displays difficulties extending from correct C:8-oxo-G in contrast to extension from incorrect A:8-oxo-G. This leads to stalling of pol δ at 8-oxo-G after incorporation of correct C. This stalling at C:8-oxo-G can be overcome by a switch from pol δ to pols λ, ß, or η, all of which are able to assist pol δ in 8-oxo-G bypass by translesion synthesis (TLS). Importantly, however, only pol λ selectively catalyzes the correct TLS past 8-oxo-G, whereas pols ß and η show no selectivity and even preferentially enhance incorrect TLS. The selectivity of pol λ to promote the correct bypass depends on its N-terminal domain. Furthermore, pol λ(-/-) mouse embryonic fibroblast extracts display reduced 8-oxo-G TLS. Finally, the correct bypass of 8-oxo-G in gapped plasmids in mouse embryonic fibroblasts and HeLa cells is promoted in the presence of pol λ. Our findings suggest that even though 8-oxo-G is not a blocking lesion per se, correct replication over 8-oxo-G is promoted by a pol switch between pols δ and λ.

Genetic requirement for mutagenesis of the G[8,5-Me]T cross-link in Escherichia coli: DNA polymerases IV and V compete for error-prone bypass.

γ-Radiation generates a variety of complex lesions in DNA, including the G[8,5-Me]T intrastrand cross-link in which C8 of guanine is covalently linked to the 5-methyl group of the 3'-thymine. We have investigated the toxicity and mutagenesis of this lesion by replicating a G[8,5-Me]T-modified plasmid in Escherichia coli with specific DNA polymerase knockouts. Viability was very low in a strain lacking pol II, pol IV, and pol V, the three SOS-inducible DNA polymerases, indicating that translesion synthesis is conducted primarily by these DNA polymerases. In the single-polymerase knockout strains, viability was the lowest in a pol V-deficient strain, which suggests that pol V is most efficient in bypassing this lesion. Most mutations were single-base substitutions or deletions, though a small population of mutants carrying two point mutations at or near the G[8,5-Me]T cross-link was also detected. Mutations in the progeny occurred at the cross-linked bases as well as at bases near the lesion site, but the mutational spectrum varied on the basis of the identity of the DNA polymerase that was knocked out. Mutation frequency was the lowest in a strain that lacked the three SOS DNA polymerases. We determined that pol V is required for most targeted G → T transversions, whereas pol IV is required for the targeted T deletions. Our results suggest that pol V and pol IV compete to carry out error-prone bypass of the G[8,5-Me]T cross-link.

DNA polymerases beta and lambda mediate overlapping and independent roles in base excision repair in mouse embryonic fibroblasts.

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol beta) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol lambda), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases lambda and beta in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol beta and pol lambda. Neutral red viability assays demonstrated that pol lambda and pol beta double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol lambda to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol lambda and pol beta interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.

Involvement of DNA polymerase beta in repairing oxidative damages induced by antitumor drug adriamycin.

Adriamycin (ADM) is a widely used antineoplastic drug. However, the increasing cellular resistance has become a serious limitation to ADM clinical application. The most important mechanism related to ADM-induced cell death is oxidative DNA damage mediated by reactive oxygen species (ROS). Base excision repair (BER) is a major pathway in the repair of DNA single strand break (SSB) and oxidized base. In this study, we firstly applied the murine embryo fibroblasts wild-type (pol ß +/+) and homozygous pol ß null cell (pol ß -/-) as a model to investigate ADM DNA-damaging effects and the molecular basis underlying these effects. Here, cellular sensitivity to ADM was examined using colorimetric assay and colony forming assay. ADM-induced cellular ROS level and the alteration of superoxide dismutase (SOD) activity were measured by commercial kits. Further, DNA strand break, chromosomal damage and gene mutation were assessed by comet assay, micronucleus test and hprt gene mutation assay, respectively. The results showed that pol ß -/- cells were more sensitive to ADM compared with pol ß +/+ cells and more severe SSB and chromosomal damage as well as higher hprt gene mutation frequency were observed in pol ß -/- cells. ROS level in pol ß -/- cells increased along with decreased activity of SOD. These results demonstrated that pol ß deficiency could enable ROS accumulation with SOD activity decrease, further elevate oxidative DNA damage, and subsequently result in SSB, chromosome cleavage as well as gene mutation, which may be partly responsible for the cytotoxicity of ADM and the hypersensitivity of pol ß -/- cells to ADM. These findings suggested that pol ß is vital for repairing oxidative damage induced by ADM.

Bioenergetic metabolites regulate base excision repair-dependent cell death in response to DNA damage.

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance [DNA polymerase beta (Polbeta) deficiency or repair inhibition] enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polbeta triggers cell death dependent on poly(ADP-ribose) (PAR) polymerase activation yet independent of PAR-mediated apoptosis-inducing factor nuclear translocation or PAR glycohydrolase, suggesting that cytotoxicity is not from PAR or PAR catabolite signaling. Cell death is rescued by the NAD(+) metabolite beta-nicotinamide mononucleotide and is synergistic with inhibition of NAD(+) biosynthesis, showing that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polbeta-deficient cells, suggesting a linkage between DNA repair, cell survival, and cellular bioenergetics.

Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy.

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment.

Ionizing radiation sensitivity of DNA polymerase lambda-deficient cells.

Ionizing radiation induces a diverse spectrum of DNA lesions, including strand breaks and oxidized bases. In mammalian cells, ionizing radiation-induced lesions are targets of non-homologous end joining, homologous recombination, and base excision repair. In vitro assays show a potential involvement of DNA polymerase lambda in non-homologous end joining and base excision repair. In this study, we investigated whether DNA polymerase lambda played a significant role in determining ionizing radiation sensitivity. Despite increased sensitivity to hydrogen peroxide, lambda-deficient mouse embryonic fibroblasts displayed equal survival after exposure to ionizing radiation compared to their wild-type counterparts. In addition, we found increased sensitivity to the topoisomerase inhibitors camptothecin and etoposide in the absence of polymerase lambda. These results do not reveal a major role for DNA polymerase lambda in determining radiosensitivity in vivo.

DNA polymerase beta is able to repair breaks in switch regions and plays an inhibitory role during immunoglobulin class switch recombination.

Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), which converts cytosines to uracils in switch (S) regions. Subsequent excision of dU by uracil DNA glycosylase (UNG) of the base excision repair (BER) pathway is required to obtain double-strand break (DSB) intermediates for CSR. Since UNG normally initiates faithful repair, it is unclear how the AID-instigated S region lesions are converted into DSBs rather than correctly repaired by BER. Normally, DNA polymerase beta (Polbeta) would replace the dC deaminated by AID, leading to correct repair of the single-strand break, thereby preventing CSR. We address the question of whether Polbeta might be specifically down-regulated during CSR or inhibited from accessing the AID-instigated lesions, or whether the numerous AID-initiated S region lesions might simply overwhelm the BER capacity. We find that nuclear Polbeta levels are induced upon activation of splenic B cells to undergo CSR. When Polbeta(-/-) B cells are activated to switch in culture, they switch slightly better to IgG2a, IgG2b, and IgG3 and have more S region DSBs and mutations than wild-type controls. We conclude that Polbeta attempts to faithfully repair S region lesions but fails to repair them all.

Decreased PARP-1 levels accelerate embryonic lethality but attenuate neuronal apoptosis in DNA polymerase beta-deficient mice.

In mammalian cells, DNA polymerase beta (Polbeta) and poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in base excision repair (BER) and single-strand break repair. Polbeta knockout mice exhibit extensive neuronal apoptosis during neurogenesis and die immediately after birth, while PARP-1 knockout mice are viable and display hypersensitivity to genotoxic agents and genomic instability. Although accumulating biochemical data show functional interactions between Polbeta and PARP-1, such interactions in the whole animal have not yet been explored. To study this, we generate Polbeta(-/-)PARP-1(-/-) double mutant mice. Here, we show that the double mutant mice exhibit a profound developmental delay and embryonic lethality at mid-gestation. Importantly, the degree of the neuronal apoptosis was dramatically reduced in PARP-1 heterozygous mice in a Polbeta null background. The reduction was well correlated with decreased levels of p53 phosphorylation at serine-18, suggesting that the apoptosis depends on the p53-mediated apoptosis pathway that is positively regulated by PARP-1. These results indicate that functional interactions between Polbeta and PARP-1 play important roles in embryonic development and neurogenesis.

DNA repair in aging rat neurons.

This laboratory, using post-mitotic rat brain neurons as a model system, has been testing the hypothesis that the inherited DNA repair potential would have profound influence on the aging process of the individual. It has been found that both single and double strand breaks in DNA accumulate in neurons with age. Since base excision repair (BER) is the pathway to effect repair of the type of DNA damage that is likely to occur in neurons, model oligo duplexes were used to assess the BER pathway. Both extension of a primer and one or four nucleotide gap repair are markedly reduced in aging neurons as compared with the young. The extension activity could be restored by supplementing the neuronal extracts with pure DNA polymerase beta (pol beta) while the restoration of gap repair needed the addition of both pol beta and DNA ligase. It thus appears that both pol beta and DNA ligase are deficient in aging neurons. We have also established a system to study the non-homologous end joining (NHEJ) mode of DNA repair in neurons. The end joining of cohesive but not of blunt or non-matching ends, is reduced with age and attempts to identify the limiting factor(s) in this case have been unsuccessful so far. These results are reviewed vis-à-vis the existing literature.

Mammary carcinogenesis in transgenic mice expressing a dominant-negative mutant of DNA polymerase beta in their mammary glands.

DNA polymerase beta (polbeta) is a major contributor to mammalian DNA damage repair through its gap-filling DNA synthesis and 5'-deoxyribose phosphate lyase activities. In this way, polbeta plays pivotal roles in the repair of oxidative DNA damage, replication, embryonic survival, neuronal development, meiosis, apoptosis and telomere function. A 36 kDa truncated polbetaDelta protein is expressed in human colorectal, breast, lung and renal carcinomas, but not in normal matched tissues. Interestingly, a binary protein-protein complex of polbetaDelta and X-ray cross-complementing group 1 acts as dominant-negative mutant. In this study, the potential tumorigenic activity of polbetaDelta was examined in nude and transgenic mouse models. Mouse embryonic fibroblasts (MEFs) expressing polbetaDelta in the absence of endogenous polbeta exhibited increased susceptibility to N-methyl-N-nitrosourea (MNU)-induced morphological transformation as compared with cells expressing wild-type (WT) polbeta. This was accompanied by reduced gap-filling DNA synthesis activity. Anchorage-independent transformed cells derived from polbetaDelta-expressing MEFs induced 100% tumor occurrence in nude mice. To support these data, we established transgenic mice expressing polbetaDelta specifically in the mammary glands from a whey acidic protein promoter-driven transgene. This is the first report of transgenic mice with tissue-specific expression of polbetaDelta. MNU-induced tumor formation was analyzed in transgenic mice expressing polbetaDelta together with endogenous WT polbeta in their mammary glands and in normal control mice expressing only WT polbeta. The latent period of tumor appearance was markedly shorter and tumor incidence was significantly higher in transgenic animals than in control animals treated under the same conditions. These results indicate that cells expressing the mutant polbetaDelta display an enhanced sensitivity to MNU that probably underlies an increased susceptibility to tumorigenesis.

Role for DNA polymerase beta in response to ionizing radiation.

Evidence for a role of DNA polymerase beta in determining radiosensitivity is conflicting. In vitro assays show an involvement of DNA polymerase beta in single strand break repair and base excision repair of oxidative damages, both products of ionizing radiation. Nevertheless the lack of DNA polymerase beta has been shown to have no effect on radiosensitivity. Here we show that mouse embryonic fibroblasts deficient in DNA polymerase beta are considerably more sensitive to ionizing radiation than wild-type cells, but only when confluent. The inhibitor methoxyamine renders abasic sites refractory to the dRP lyase activity of DNA polymerase beta. Methoxyamine did not significantly change radiosensitivity of wild-type fibroblasts in log phase. However, DNA polymerase beta deficient cells in log phase were radiosensitized by methoxyamine. Alkaline comet assays confirmed repair inhibition of ionizing radiation induced damage by methoxyamine in these cells, indicating both the existence of a polymerase beta-dependent long patch pathway and the involvement of another methoxyamine sensitive process, implying the participation of a second short patch polymerase(s) other than DNA polymerase beta. This is the first evidence of a role for DNA polymerase beta in radiosensitivity in vivo.

The polymerases for V(D)J recombination.

DNA transactions of a wide variety generally require three major types of enzymatic activities: nucleases, polymerases, and ligases. V(D)J recombination is no exception. In this issue, Bertocci et al. (2006) have provided new insight by generating mice deficient in one or more of the polymerases.

Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo.

DNA polymerases mu (pol mu), lambda (pol lambda), and terminal deoxynucleotidyltransferase (TdT) are enzymes of the pol X family that share homology in sequence and functional domain organization. We showed previously that pol mu participates in light chain but surprisingly not heavy chain gene rearrangement. We show here that immunoglobulin heavy chain junctions from pol lambda-deficient animals have shorter length with normal N-additions, thus indicating that pol lambda is recruited during heavy chain rearrangement at a step that precedes the action of TdT. In contrast to previous in vitro studies, analysis of animals with combined inactivation of these enzymes revealed no overlapping or compensatory activities for V(D)J recombination between pol mu, pol lambda, and TdT. This complex usage of polymerases with distinct catalytic specificities may correspond to the specific function that the third hypervariable region assumes for each immunoglobulin chain, with pol lambda maintaining a large heavy chain junctional heterogeneity and pol mu ensuring a restricted light chain junctional variability.

DNA polymerase beta promotes recruitment of DNA ligase III alpha-XRCC1 to sites of base excision repair.

Base excision repair is a major pathway for the removal of simple lesions in DNA including base damage and base loss (abasic site). Base excision repair requires the coordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. Using a protein-DNA cross-linking assay during repair in human whole cell extracts, we monitored proteins involved in the initial steps of repair of a substrate containing a site-specific abasic site to address the molecular events following incision of the abasic site by AP endonuclease. We find that after dissociation of AP endonuclease from the incised abasic site, both DNA polymerase beta (Pol beta) and the DNA ligase IIIalpha-XRCC1 heterodimer efficiently bind/cross-link to the substrate DNA. We also find that the cross-linking efficacy of the DNA ligase IIIalpha-XRCC1 heterodimer was decreased about 2-fold in the Pol beta-deficient cell extract but was rescued by addition of purified wild type but not a mutant Pol beta protein that does not interact with the DNA ligase IIIalpha-XRCC1 heterodimer. We further demonstrate that Pol beta and the DNA ligase IIIalpha-XRCC1 heterodimer are present at equimolar concentrations in whole cell extracts and that Pol beta has a 7-fold higher affinity to the incised abasic site containing substrate than DNA ligase IIIalpha. Using gel filtration of whole cell extracts prepared at physiological salt conditions (0.15 M NaCl), we find no evidence for a stable preexisting complex of DNA Pol beta with the DNA ligase IIIalpha-XRCC1 heterodimer. Taken together, these data suggest that following incision by AP endonuclease, DNA Pol beta recognizes and binds to the incised abasic site and promotes recruitment of the DNA ligase IIIalpha-XRCC1 heterodimer through its interaction with XRCC1.

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.

DNA polymerase lambda protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair.

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.