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DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate, restart of stalled forks, and replication checkpoint efficiency. Pol κ is also required for the stabilization of stalled forks, although the mechanisms are poorly understood. In this study, we unveiled an unexpected role for Pol κ in controlling the stability and abundance of checkpoint kinase 1 (Chk1), an important actor for the replication checkpoint and fork stabilization. We found that loss of Pol κ decreased the Chk1 protein level in the nuclei of four human cell lines. Pol κ and not the other Y family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation. Importantly, we also observed that the fork restart defects observed in Pol κ-depleted cells could be overcome by the reexpression of Chk1. Strikingly, this new function of Pol κ does not require its catalytic activity. We propose that Pol κ could contribute to the protection of stalled forks through Chk1 stability.

DNA polymerase kappa (Pol κ) has been well documented thus far for its specialized DNA synthesis activity during translesion replication, progression of replication forks through regions difficult to replicate and replication checkpoint at stalled forks.Here we unveiled an unexpected role for Pol κ in controlling the stability and abundance of Chk1, the major mediator of the replication checkpoint. We found that loss of Pol κ decreased the Chk1 protein level in the nucleus of four human cell lines. Pol κ and not the other Y‐family polymerase members is required to maintain the Chk1 protein pool all along the cell cycle. We showed that Pol κ depletion affected the protein stability of Chk1 and protected it from proteasome degradation and the replication recovery defects observed in Pol κ-depleted cells could be overcome by the re-expression of Chk1. Importantly, this new function of Pol κ does not require its catalytic activity, revealing that in addition to its known roles in the replication process, Pol κ can contribute to the maintenance of genome stability independently of its DNA synthesis activity.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially N2-dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the POLK gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.

DNA polymerase kappa (POLK), one of the specialized Y family DNA polymerases, functions in translesion synthesis and is suggested to be related with cancers. Single nucleotide polymorphisms (SNPs) in specialized DNA polymerases have been demonstrated to be associated with cancer risk. To evaluate the association of two common POLK variants (rs3213801 C>T and rs5744533 C>T) with glioma, we conducted a case‐control study and genotyped these two POLK variants in 605 patients and 1300 healthy controls. The association analysis revealed no significant correlations were observed between these two POLK SNPs and glioma risk. However, the POLK rs3213801 CT genotype was found to be higher in older glioma patients (≥40) than in younger patients (P = .026). Compared with patients harboring the CC genotype, the frequencies of POLK rs5744533 CT and CT+TT genotypes were increased in patients with lower World Health Organization (WHO) grade glioma (P = .028, 0.044, respectively). According to Kaplan‐Meier analysis and log‐rank tests, POLK SNPs were not correlated with either the overall survival or progression‐free survival. Nevertheless, multivariate analysis revealed that the age (≥40) could increase the risk of death in glioma patients (P, In this study, we found DNA polymerase kappa variants rs3213801 and rs5744533 are not associated with glioma risk and prognosis, but they are likely to be associated with certain glioma characteristics, such as age and World Health Organization grade. Meanwhile, the age, surgery types, and chemotherapy could be independent prognostic factors in glioma.

Genotoxic carcinogens are regulated under the policy that there is no threshold or safe dose. It has been pointed out, however, that self-defense mechanisms, such as detoxification, DNA repair, and error-free translesion synthesis, may protect chromosome DNA from genotoxic insults, thereby constituting practical threshold. In this study, we examined dose responses of chromosome aberrations induced by three oxidative genotoxins, that is, hydrogen peroxide (H2 O2 ), menadione and paraquat, with or without DNA polymerase kappa (Polκ) activities and mismatch repair capacities in human cells. Polκ is involved in translesion synthesis across DNA damage and mismatch repair is responsible for correction of base-base mismatch in DNA. Polκ activity of the cells was inactivated either by point mutations in the catalytically essential amino acids (catalytically dead or CD) or by deletion of the POLK gene (knockout or KO). In the absence of mismatch repair, frequencies of chromosome aberrations induced by H2 O2 and menadione were not significantly different among CD, KO, and the wild type (WT) cells. In the presence of mismatch repair, however, cytotoxicity and clastogenicity were enhanced and Polκ modulated the sensitivity of the cells. No-observed-genotoxic-effect-levels (NOGELs) for H2 O2 and menadione were CD = KO < WT cells. In contrast, the sensitivities of the cells to paraquat were not significantly affected by the status of mismatch repair or Polκ activity. The results suggest that mismatch repair and Polκ coordinately modulate NOGELs for the clastogenicity of H2 O2 and menadione and also that DNA lesion(s) responsible for paraquat-induced chromosome aberrations are different from those induced by H2 O2 and menadione. Environ. Mol. Mutagen. 61:193-199, 2020. © 2019 Wiley Periodicals, Inc.

Drug Resistance DNA polymerase kappa (Polκ) is an error-prone polymerase that is overexpressed in some tumors. Temprine et al. found that Polκ facilitates tumor cell survival in response to oncogenic mutations, targeted kinase inhibition, oxidative stress, or starvation (see the Focus by Sweasy). These cellular stresses shifted Polκ from its largely cytoplasmic distribution to a nuclear localization in various tumor cell types. However, mutagenesis was not increased. Expression of a constitutively nuclear Polκ enhanced the resistance of melanoma cells to the cancer drug vemurafenib. Sci. Signal. 13 , eaau1453, eabb2934 (2020).

In vitro studies indicate that DNA polymerase kappa (Polκ) is able to accurately and efficiently perform DNA synthesis using templates containing various types of DNA damage, including benzo[a]pyrene (BP)-induced N2 -deoxyguanosine adducts. In this study, we examined sensitivity of inactivated Polk knock-in (Polk-/- ) mice to BP carcinogenicity in the colon by administering an oral dose of BP plus dextran sulfate sodium (DSS), an inflammation causing promoter of carcinogenesis. Although colon cancer was successfully induced by BP plus DSS, there was no significant difference in tumor incidence or multiplicity between Polk-/- and Polk+/+ mice. Malignant lymphoma was induced in thymus by the treatment only in Polk-/- mice, but it lacked statistical significance. Mutant frequencies (MFs) in the gpt reporter gene were strongly enhanced in colon; almost to the same extent in both types of mice. Micronucleus formation in bone marrow at the high dose of BP and DNA adducts in colon and lung was not significantly different between two types of mice. Surprisingly, however, Polk-/- mice exhibited significantly higher MFs in colon and lung than did Polk+/+ mice when they were treated with DSS alone. The most prominent mutation induced by DSS treatment was G:C to C:G transversion, whose specific MF in proximal colon was 30 times higher in Polk-/- than in Polk+/+ mice. DSS alone did not enhance MF at all in Polk+/+ mice. The results indicate that Polκ does not suppress BP-induced mutagenesis and carcinogenesis in the colon, but counteracts inflammation-induced mutagenesis in multiple organs. Environ. Mol. Mutagen. 60:320-330, 2019. © 2019 Wiley Periodicals, Inc.

DNA polymerase kappa is a Y-family polymerase that participates to bypass the damaged DNA known as translesion synthesis (TLS) polymerase. Higher frequency of mutations in DNA polymerase kappa (POLK) recently been reported in prostate cancer. We sequenced entire exons of the POLK gene on genomic DNA from 40 prostate cancers and matched normal samples. We identified that 28% of patients have somatic mutations in the POLK gene of the prostate tumors. Mutations in these prostate cancers have somatic mutation spectra, which are dominated by C-to-T transitions. In the current study, we further investigate the effect of p.E29K, p.G154E, p.F155S, p.E430K, p.L442F, and p.E449K mutations on the biochemical properties of the polymerase in vitro, using TLS assay and nucleotide incorporation fidelity, following site-directed mutagenesis bacterial expression, and purification of the respective polymerase variants. We report that following missense mutations p.E29K, p.G154E, p.F155S, p.E430K, and p.L442F significantly diminished the catalytic efficiencies of POLK with regard to the lesion bypass (AP site). POLK variants show extraordinarily low fidelity by misincorporating T, C, and G as compared to wild-type variants. Taken together, these results suggest that interfering with normal polymerase kappa function by these mutations may be involved in prostate carcinogenesis.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk + ) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk + mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N2-deoxyguanine (BPDE-N2-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (−)- and (+)-trans-anti-BPDE-N2-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/−), F171A (POLK F171A/−) or lack expression of Pol κ (POLK−/−) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (−)- or (+)-trans-anti-BPDE-N2-dG in the supF gene. The frequencies of mutations were in the order of POLK−/− > POLK+/− > POLK F171A/− both in (−)- and (+)-trans-anti-BPDE-N2-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N2-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

DNA polymerase kappa (Polκ) bypasses planar polycyclic N2-guanine adducts in an error-free manner. Cholesterol derivatives may interact with DNA to form similarly bulky lesions. In accordance, these studies examined whether increased mutagenesis of DNA accompanies hypercholesterolemia in Polk−/− mice. These mice also carried apoE gene knockouts to ensure increased levels of plasma cholesterol following exposure to a high cholesterol diet. The mice carried a reporter transgene (the λ-phage cII gene) for subsequent quantitative analysis of mutagenesis in various tissues. We observed significantly increased mutation frequencies in several organs of apoE−/−Polk−/− mice following a high cholesterol diet, compared to those remaining on a standard diet. Regardless of dietary regime, the mutation frequency in many organs was significantly higher in apoE−/−Polk−/− than in apoE−/−Polk+/+ mice. As expected for polycyclic guanine adducts, the mutations mainly consisted of G:C transversions. The life expectancy of apoE−/−Polk−/− mice maintained on a high cholesterol diet was reduced compared to apoE−/−Polk+/+ mice. Overall, this study demonstrates a role for Polκ in bypass of cholesterol-induced guanine lesions.

The Y-family of DNA polymerases support of translesion DNA synthesis (TLS) associated with stalled DNA replication by DNA damage. Recently, a number of studies suggest that some specialized TLS polymerases also support other aspects of DNA metabolism beyond TLS in vivo. Here we show that mouse polymerase kappa (Polκ) could accumulate at laser-induced sites of damage in vivo resembling polymerases eta and iota. The recruitment was mediated through Polκ C-terminus which contains the PCNA-interacting peptide, ubiquitin zinc finger motif 2 and nuclear localization signal. Interestingly, this recruitment was significantly reduced in MSH2-deficient LoVo cells and Rad18-depleted cells. We further observed that Polκ-deficient mouse embryo fibroblasts were abnormally sensitive to H2O2 treatment and displayed defects in both single-strand break repair and double-strand break repair. We speculate that Polκ may have an important role in strand break repair following oxidative stress in vivo.

DNA polymerase kappa (Pol kappa) is a low-fidelity polymerase that has the ability to bypass several types of lesions. The biological role of this enzyme, a member of the DinB subfamily of Y-family DNA polymerases, has remained elusive. In this report, we studied one of the two copies of Pol kappa from the protozoan Trypanosoma cruzi (TcPol kappa). The role of this TcPol kappa copy was investigated by analysing its subcellular localization, its activities in vitro, and performing experiments with parasites that overexpress this polymerase. The TcPOLK sequence has the N-terminal extension which is present only in eukaryotic DinB members, but its C-terminal region is more similar to prokaryotic and archaeal counterparts since it lacks C(2)HC motifs and PCNA interaction domain. Our results indicate that in contrast to its previously described orthologues, this polymerase is localized to mitochondria. The overexpression of TcPOLK increases T. cruzi resistance to hydrogen peroxide, and in vitro polymerization assays revealed that TcPol kappa efficiently bypasses 8-oxoguanine lesions. Remarkably, our results also demonstrate that the DinB subfamily of polymerases can participate in homologous recombination, based on our findings that TcPol kappa increases T. cruzi resistance to high doses of gamma irradiation and zeocin and can catalyse DNA synthesis within recombination intermediates.

Microsatellites are ubiquitously present in eukaryotic genomes and are implicated as positive factors in evolution. At the nucleotide level, microsatellites undergo slippage events that alter allele length and base changes that interrupt the repetitive tract. We examined DNA polymerase errors within a [T](11) microsatellite using an in vitro assay that preferentially detects mutations other than unit changes. We observed that human DNA polymerase kappa (Pol kappa) inserts dGMP and dCMP within the [T](11) mononucleotide repeat, producing an interrupted 12-bp allele. Polymerase beta produced such interruptions at a lower frequency. These data demonstrate that DNA polymerases are capable of directly producing base interruptions within microsatellites. At the molecular level, expanded microsatellites have been implicated in DNA replication fork stalling. Using an in vitro primer extension assay, we observed sequence-specific synthesis termination by DNA polymerases within mononucleotides. Quantitatively, intense, polar pausing was observed for both pol kappa and polymerase alpha-primase within a [T](11) allele. A mechanism is proposed in which pausing results from DNA bending within the duplex stem of the nascent DNA. Our data support the concept of a microsatellite life-cycle, and are consistent with the models in which DNA sequence or secondary structures contributes to non-uniform rates of replication fork progression.

DNA replication is a major event for cells which allow the faithful duplication of genetic material. It is a critical step of cell cycle, because replication forks encounters frequently naturals barriers (non B-DNA structures), exogenous barriers (chemicals agents), or endogenous barriers (oxydatives lesions). These different barriers can be at the origin of chromosomes breaks and lead to genetic instability. To overcome the stalled forks, cells have evolved two major mechanisms: the induction of ATR replication checkpoint pathway and the recruitment of specialized DNA polymerase to perform the translesion synthesis. This two pathways are essential to maintain genomic stability. Human DNA polymerase Kappa (pol Kappa), the most conserved specialized DNA polymerase, is best known to participate to translesion synthesis. Recently, we have shown that pol kappa has a crucial role in the S-phase checkpoint activation. Indeed, pol Kappa is implicated in the synthesis of short DNA intermediates at the stalled forks, facilitating the recruitment of 9-1-1 clamp, and is required for an optimal phosphorylation of Chk1, the main effector of the S-phase checkpoint. Durant my PhD thesis, I explored the molecular mechanisms underlying this newly identified role. We have constructed several pol kappa mutants, and we have observed that for the mutation in the PCNA binding domain impeded pol kappa to form foci in response to replication stress. We also showed the requirement of this domain for pol Kappa recruitment on chromatin. By different experimental approaches, we have described a complex in which pol Kappa interacts with Rad9 and Chk1, two proteins required for the S-phase checkpoint activation. The Chk1 phosphorylation defect observed in absence of Kappa could also be the consequence of the Chk1 protein level decreased in the nucleus, meaning a potential common regulation between pol Kappa and Chk1. Based on this observation, we have studied how pol Kappa is regulated upon a replication stress and like Chk1, pol Kappa seems to be regulated by ubiquitination. We focused our attention on USP7 an ubiquitin hydrolase known to regulate Chk1. We have demonstrated an interaction between pol Kappa and USP7, which is stimulated after replication stress. Moreover, USP7 depletion leads to a decrease of pol Kappa level in the nucleus, suggesting that de-ubiquination of pol Kappa could to be a prerequisite for its checkpoint function and its stability.; La réplication de l'ADN est un évènement majeur pour la cellule car elle permet la duplication fidèle du matériel génétique. Il s'agit d'une étape critique du cycle cellulaire, car les fourches de réplication rencontrent fréquemment des barrières naturelles ou des lésions d'origine endogènes (lésions oxydatives) ou exogènes (agents physiques ou chimiques), sources de cassures chromosomiques et donc d'instabilité génétique. Une des réponses à ces fourches bloquées est l'activation du point de contrôle (checkpoint) de la phase S du cycle cellulaire. Nous avons montré que l'ADN polymérase Kappa (pol Kappa), polymérase dite translesionnelle en raison de ses capacité à franchir des lésions sur l'ADN, s'avère être aussi un acteur du point de contrôle de phase S. En effet, la déplétion de pol Kappa par ARN interférence dans différentes lignées cellulaires ou par immunodépletion d'un extrait de Xénope, entraîne un défaut de phosphorylation de Chk1. Pol Kappa est impliquée dans la synthèse de brins naissant d'ADN au niveau des fourches bloquées, ce qui facilite le recrutement du complexe 9-1-1 composé des protéines Rad9, Rad1 et Hus1et permet alors, une activation correcte du checkpoint de phase S. Afin de décrypter le rôle de pol kappa, nous avons construits différents mutants et nous avons analysé leur capacité à former des foyers, à être recrutés à la chromatine et à interagir avec différents partenaires dans des conditions d'activation du point de contrôle de phase S. Nous avons pu constater que le mutant du domaine d'interaction à PCNA était incapable de former des foci foyers ?. Nous avons ensuite observé, qu'en condition de stress réplicatif, pol Kappa était recruté à la chromatine grâce à son domaine d'interaction à PCNA et par différentes approches biochimiques, nous avons pu voir que pol kappa interagissait avec Rad9 et Chk1. Nous avons également mis en évidence que le défaut d'activation de Chk1 en l'absence de pol kappa reflétait d'une diminution de son taux dans le noyau, suggérant une régulation commune entre Chk1 et pol Kappa. En effet, nous avons observé que pol Kappa, comme Chk1, était régulés par l'ubiquitine hydrolase USP7. En effet, l'interaction entre pol Kappa et USP7 est augmentée en condition de stress. Nous avons pu voir, qu'à l'instar de Chk1, l'absence de USP7 entrainait une baisse du niveau de pol kappa dans le noyau. Ainsi nous proposons qu'en réponse à un stress réplicatif, pol Kappa et Chk1 soient stabilisés via leur dé-ubiquitination par USP7, permettant leur recrutement à la chromatine et une activation correcte du checkpoint de phase S. Parallèlement à ces travaux, des publications récentes montrent une implication possible de pol Kappa au niveau des séquences répétées. Nous avons pu mettre en évidence une interaction entre pol Kappa et Cenpb, protéine centromérique reconnaissant une séquence de 17 paires de bases dans l'ADN a-satellite. Ces résultats préliminaires suggèrent que le rôle de pol Kappa dans le checkpoint de phase S s'adresse notamment aux régions d'hétérochromatine. L'ensemble des résultats obtenus montre l'importance de pol Kappa dans le maintien de la stabilité génomique, par son rôle dans le checkpoint de phase S, et par son implication dans la régulation de Chk1 en condition de stress réplicatif.

DNA replication is a major event for cells which allow the faithful duplication of genetic material. It is a critical step of cell cycle, because replication forks encounters frequently naturals barriers (non B-DNA structures), exogenous barriers (chemicals agents), or endogenous barriers (oxydatives lesions). These different barriers can be at the origin of chromosomes breaks and lead to genetic instability. To overcome the stalled forks, cells have evolved two major mechanisms: the induction of ATR replication checkpoint pathway and the recruitment of specialized DNA polymerase to perform the translesion synthesis. This two pathways are essential to maintain genomic stability. Human DNA polymerase Kappa (pol Kappa), the most conserved specialized DNA polymerase, is best known to participate to translesion synthesis. Recently, we have shown that pol kappa has a crucial role in the S-phase checkpoint activation. Indeed, pol Kappa is implicated in the synthesis of short DNA intermediates at the stalled forks, facilitating the recruitment of 9-1-1 clamp, and is required for an optimal phosphorylation of Chk1, the main effector of the S-phase checkpoint. Durant my PhD thesis, I explored the molecular mechanisms underlying this newly identified role. We have constructed several pol kappa mutants, and we have observed that for the mutation in the PCNA binding domain impeded pol kappa to form foci in response to replication stress. We also showed the requirement of this domain for pol Kappa recruitment on chromatin. By different experimental approaches, we have described a complex in which pol Kappa interacts with Rad9 and Chk1, two proteins required for the S-phase checkpoint activation. The Chk1 phosphorylation defect observed in absence of Kappa could also be the consequence of the Chk1 protein level decreased in the nucleus, meaning a potential common regulation between pol Kappa and Chk1. Based on this observation, we have studied how pol Kappa is regulated upon a replication stress and like Chk1, pol Kappa seems to be regulated by ubiquitination. We focused our attention on USP7 an ubiquitin hydrolase known to regulate Chk1. We have demonstrated an interaction between pol Kappa and USP7, which is stimulated after replication stress. Moreover, USP7 depletion leads to a decrease of pol Kappa level in the nucleus, suggesting that de-ubiquination of pol Kappa could to be a prerequisite for its checkpoint function and its stability., La réplication de l'ADN est un évènement majeur pour la cellule car elle permet la duplication fidèle du matériel génétique. Il s'agit d'une étape critique du cycle cellulaire, car les fourches de réplication rencontrent fréquemment des barrières naturelles ou des lésions d'origine endogènes (lésions oxydatives) ou exogènes (agents physiques ou chimiques), sources de cassures chromosomiques et donc d'instabilité génétique. Une des réponses à ces fourches bloquées est l'activation du point de contrôle (checkpoint) de la phase S du cycle cellulaire. Nous avons montré que l'ADN polymérase Kappa (pol Kappa), polymérase dite translesionnelle en raison de ses capacité à franchir des lésions sur l'ADN, s'avère être aussi un acteur du point de contrôle de phase S. En effet, la déplétion de pol Kappa par ARN interférence dans différentes lignées cellulaires ou par immunodépletion d'un extrait de Xénope, entraîne un défaut de phosphorylation de Chk1. Pol Kappa est impliquée dans la synthèse de brins naissant d'ADN au niveau des fourches bloquées, ce qui facilite le recrutement du complexe 9-1-1 composé des protéines Rad9, Rad1 et Hus1et permet alors, une activation correcte du checkpoint de phase S. Afin de décrypter le rôle de pol kappa, nous avons construits différents mutants et nous avons analysé leur capacité à former des foyers, à être recrutés à la chromatine et à interagir avec différents partenaires dans des conditions d'activation du point de contrôle de phase S. Nous avons pu constater que le mutant du domaine d'interaction à PCNA était incapable de former des foci foyers ?. Nous avons ensuite observé, qu'en condition de stress réplicatif, pol Kappa était recruté à la chromatine grâce à son domaine d'interaction à PCNA et par différentes approches biochimiques, nous avons pu voir que pol kappa interagissait avec Rad9 et Chk1. Nous avons également mis en évidence que le défaut d'activation de Chk1 en l'absence de pol kappa reflétait d'une diminution de son taux dans le noyau, suggérant une régulation commune entre Chk1 et pol Kappa. En effet, nous avons observé que pol Kappa, comme Chk1, était régulés par l'ubiquitine hydrolase USP7. En effet, l'interaction entre pol Kappa et USP7 est augmentée en condition de stress. Nous avons pu voir, qu'à l'instar de Chk1, l'absence de USP7 entrainait une baisse du niveau de pol kappa dans le noyau. Ainsi nous proposons qu'en réponse à un stress réplicatif, pol Kappa et Chk1 soient stabilisés via leur dé-ubiquitination par USP7, permettant leur recrutement à la chromatine et une activation correcte du checkpoint de phase S. Parallèlement à ces travaux, des publications récentes montrent une implication possible de pol Kappa au niveau des séquences répétées. Nous avons pu mettre en évidence une interaction entre pol Kappa et Cenpb, protéine centromérique reconnaissant une séquence de 17 paires de bases dans l'ADN a-satellite. Ces résultats préliminaires suggèrent que le rôle de pol Kappa dans le checkpoint de phase S s'adresse notamment aux régions d'hétérochromatine. L'ensemble des résultats obtenus montre l'importance de pol Kappa dans le maintien de la stabilité génomique, par son rôle dans le checkpoint de phase S, et par son implication dans la régulation de Chk1 en condition de stress réplicatif.

International audience; Mutations at A/T bases within immunoglobulin genes have been shown to be generated by a repair pathway involving the DNA-binding moiety of the mismatch repair complex constituted by the MSH2-MSH6 proteins, together with DNA polymerase eta (pol eta). However, residual A/T mutagenesis is still observed upon inactivation in the mouse of each of these factors, suggesting that the panel of activities involved might be more complex. We reported previously (Delbos, F., A. De Smet, A. Faili, S. Aoufouchi, J.-C. Weill, and C.-A. Reynaud. 2005. J. Exp. Med. 201:1191-1196) that residual A/T mutagenesis in pol eta-deficient mice was likely contributed by another enzyme not normally involved in hypermutation, DNA polymerase kappa, which is mobilized in the absence of the normal polymerase partner. We report the complete absence of A/T mutations in MSH2-pol eta double-deficient mice, thus indicating that the residual A/T mutagenesis in MSH2-deficient mice is contributed by pol eta, now recruited by uracil N-glycosylase, the second DNA repair pathway involved in hypermutation. We propose that this particular recruitment of pol eta corresponds to a profound modification of the function of uracil glycosylase in the absence of the mismatch repair complex, suggesting that MSH2-MSH6 actively prevent uracil glycosylase from error-free repair during hypermutation. pol eta thus appears to be the sole contributor of A/T mutations in the normal physiological context.

DNA polymerase beta (polbeta), a member of the X family of DNA polymerases, is the major polymerase in the base excision repair pathway. Using in vitro selection, we obtained RNA aptamers for polbeta from a variable pool of 8 x 10(12) individual RNA sequences containing 30 random nucleotides. A total of 60 individual clones selected after seven rounds were screened for the ability to inhibit polbeta activity. All of the inhibitory aptamers analyzed have a predicted tri-lobed structure. Gel mobility shift assays demonstrate that the aptamers can displace the DNA substrate from the polbeta active site. Inhibition by the aptamers is not polymerase specific; inhibitors of polbeta also inhibited DNA polymerase kappa, a Y-family DNA polymerase. However, the RNA aptamers did not inhibit the Klenow fragment of DNA polymerase I and only had a minor effect on RB69 DNA polymerase activity. Polbeta and kappa, despite sharing little sequence similarity and belonging to different DNA polymerase families, have similarly open active sites and relatively few interactions with their DNA substrates. This may allow the aptamers to bind and inhibit polymerase activity. RNA aptamers with inhibitory properties may be useful in modulating DNA polymerase activity in cells.

The Goodpasture antigen-binding protein, GPBP, is a serine/threonine kinase whose relative expression increases in autoimmune processes. Tumor necrosis factor (TNF) is a pro-inflammatory cytokine implicated in autoimmune pathogenesis. Here we show that COL4A3BP, the gene encoding GPBP, maps head-to-head with POLK, the gene encoding for DNA polymerase kappa (pol kappa), and shares with it a 140-bp promoter containing a Sp1 site, a TATA-like element, and a nuclear factor kappa B (NFkappaB)-like site. These three elements cooperate in the assembly of a bidirectional transcription complex containing abundant Sp1 and little NFkappaB that is more efficient in the POLK direction. Tumour necrosis factor cell induction is associated with Sp1 release, NFkappaB recruitment and assembly of a complex comparatively more efficient in the COL4A3BP direction. This is accomplished by competitive binding of Sp1 and NFkappaB to a DNA element encompassing a NFkappaB-like site that is pivotal for the 140-bp promoter to function. Consistently, a murine homologous DNA region, which contains the Sp1 site and the TATA-like element but is devoid of the NFkappaB-like site, does not show transcriptional activity in transient gene expression assays. Our findings identify a human-specific TNF-responsive transcriptional unit that locates GPBP in the signalling cascade of TNF and substantiates previous observations, which independently related TNF and GPBP with human autoimmunity.

Replication across unrepaired DNA lesions in mammalian cells is effected primarily by specialized, low fidelity DNA polymerases. We studied translesion DNA synthesis (TLS) across a benzo[a]pyrene-guanine (BP-G) adduct, a major mutagenic DNA lesion generated by tobacco smoke. This was done using a quantitative assay that measures TLS indirectly, by measuring the recovery of gapped plasmids transfected into cultured mammalian cells. Analysis of PolK(+/+) mouse embryo fibroblasts (MEFs) showed that TLS across the BP-G adduct occurred with an efficiency of 48 +/- 4%, which is an order of magnitude higher than in Escherichia coli. In PolK(-/-) MEFs, bypass was 16 +/- 1%, suggesting that at least two-thirds of the BP-G adducts in MEFs were bypassed exclusively by polymerase kappa (polkappa). In contrast, poleta was not required for bypass across BP-G in a human XP-V cell line. Analysis of misinsertion specificity across BP-G revealed that bypass was more error-prone in MEFs lacking polkappa. Expression of polkappa from a plasmid introduced into PolK(-/-) MEFs restored both the extent and fidelity of bypass across BP-G. Polkappa was not required for bypass of a synthetic abasic site. In vitro analysis demonstrated efficient bypass across BP-G by both polkappa and poleta, suggesting that the biological role of polkappa in TLS across BP-G is due to regulation of TLS and not due to an exclusive ability to bypass this lesion. These results indicate that BP-G is bypassed in mammalian cells with relatively high efficiency and that polkappa bypasses BP-G in vivo with higher efficiency and higher accuracy than other DNA polymerases.

A recently discovered group of novel polymerases are characterized by significantly reduced fidelity of DNA synthesis in vitro. This feature is consistent with the relaxed fidelity required for the replicative bypass of various types of base damage that frequently block high fidelity replicative polymerases. The present studies demonstrate that the specialized DNA polymerase kappa (polkappa) is uniquely and preferentially expressed in the adrenal cortex and testis of the mouse, as well as in a variety of other tissues. The adrenal cortex is the sole site of detectable expression of the Polkappa gene in mouse embryos. This adrenal expression pattern is consistent with a requirement for polkappa for the replicative bypass of DNA base damage generated during steroid biosynthesis. The expression pattern of polkappa in the testis is specific for particular stages of spermatogenesis and is distinct from the expression pattern of several other low fidelity DNA polymerases that are also expressed during spermatogenesis. The mouse (but not the human) Polkappa gene is primarily regulated by the p53 gene and is upregulated in response to exposure to various DNA-damaging agents in a p53-dependent manner.

Single nucleotide instability (SNI), an increase in spontaneous point mutation rates (MRs) without involvement of microsatellite instability, is present in rat mammary carcinoma cell lines and human breast cancer cell lines. A:T to C:G transversions, which are generally rare, were frequently observed in two rat mammary carcinoma cell lines and in their primary carcinomas, and were considered to be related to the molecular mechanism of SNI. In this study, two known molecular mechanisms that cause increases of A:T to C:G transversions, inactivation of the MutT mammalian homologue (Mth1) gene and overexpression of the DNA polymerase k (Pol k) gene, were analyzed in two rat mammary carcinoma cell lines and 11 rat primary carcinomas. PCR-SSCP analysis revealed no mutations in the entire Mth1 coding region. Quantitative real-time RT-PCR analysis showed that Mth1 mRNA expression was slightly, but significantly, increased in the primary carcinomas (P = 0.001 using GAPDH for normalization, and P = 0.002 using histone H4, t-test), contrary to our expectation, and was decreased to 1 / 2 in the cell lines. The expression of Pol k, which is known to be error-prone with frequent A:T to C:G transversions, was rather decreased in the cell lines and primary carcinomas. Inactivation of Mth1 and overexpression of Pol k were unlikely to have caused SNI in the two rat mammary carcinoma cell lines with a high frequency of A:T to C:G transversions, and searching for other unknown molecular mechanisms is important.

Several recently discovered human DNA polymerases are associated with translesion synthesis past DNA adducts. These include human DNA polymerase kappa (pol kappa), a homologue of Escherichia coli pol IV, which enhances the frequency of spontaneous mutation. Using a truncated form of pol kappa (pol kappa Delta C), translesion synthesis past dG-(+)- or dG-(-)-anti-N(2)-BPDE (7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene) adducts was explored. Site-specifically-modified oligodeoxynucleotides containing a single stereoisomeric dG-N(2)-BPDE lesion were used as DNA templates for primer extension reactions catalyzed by pol kappa Delta C. Primer extension was retarded one base prior to the dG-N(2)-BPDE lesion; when incubated for longer times or with higher concentration of enzyme, full primer extension was observed. Quantitative analysis of fully extended products showed preferential incorporation of dCMP, the correct base, opposite all four stereoisomeric dG-N(2)-BPDE lesions. (+)-trans-dG-N(2)-BPDE, a major BPDE-DNA adduct, promoted small amounts of dTMP, dAMP, and dGMP misincorporation opposite the lesion (total 2.7% of the starting primers) and deletions (1.1%). Although (+)-cis-dG-N(2)-BPDE was most effective in blocking translesion synthesis, its miscoding properties were similar to other dG-N(2)-BPDE isomers. Steady-state kinetic data indicate that dCMP is efficiently inserted opposite all dG-N(2)-BPDE adducts and extended past these lesions. The relative frequency of translesion synthesis (F(ins) x F(ext)) of dC.dG-N(2)-BPDE pairs was 2-6 orders of magnitude higher than that of other mismatched pairs. Pol kappa may play an important role in translesion synthesis by incorporating preferentially the correct base opposite dG-N(2)-BPDE. Its relatively low contribution to mutagenicity suggests that other newly discovered DNA polymerase(s) may be involved in mutagenic events attributed to dG-N(2)-BPDE adducts in human cells.

Backgrounds Human polκ is a newly identified low-fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene-adduct in an error-prone manner, it bypasses benzo[a]pyrene-N2-dG lesions in a mostly error-free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand-activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin. Results We examined promoter structures of the human POLK and mouse Polk genes to study how their expressions are regulated. The mouse Polk gene is developmentally regulated in testis and utilizes two transcription start sites during spermatogenesis, while it utilizes only one site in tissues other than testis. Both of the mouse Polk and the human POLK genes have two AhR-binding sites in the promoter regions and the expression of the mouse Polk gene is indeed enhanced upon AhR-activation. Conclusions The AhR activation increases expression of the mouse Polk gene and probably the human POLK gene, the product of which bypasses benzo[a]pyrene-N2-dG lesions in a mostly accurate manner. Thus, polκ seems to function to reduce mutagenesis at benzo[a]pyrene-adducts, although it may also have a role related to spermatogenesis.

Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of DNA polymerase kappa (Polkappa(-/-)) deficient mice. The spontaneous mutation rate in homozygous Polkappa(-/-) males was significantly higher than in isogenic wild-type mice (Polkappa(+/+)), but the ESTR mutation spectrum in Polkappa(-/-) animals did not differ from that in Polkappa(+/+) males. We suggest that compromised translesion synthesis in Polkappa(-/-) mice may result in replication fork pausing which, in turn, may affect ESTR mutation rate.

DNA polymerases are responsible for ensuring stability of the genome and avoiding genotoxicity caused by a variety of factors during DNA replication. Consequently, these proteins have been associated with an increased cancer risk. DNA polymerase kappa (POLK) is a specialized DNA polymerase involved in translesion DNA synthesis (TLS) that allows DNA synthesis over the damaged DNA. Recently, some studies investigated relationships between POLK polymorphisms and cancer risk, but the role of POLK genetic variants in breast cancer (BC) remains to be defined. In this study, we aimed to evaluate the effects of POLK polymorphisms on BC risk. We used the Sequenom MassARRAY method to genotype 3 single nucleotide polymorphisms (SNPs) in POLK (rs3213801, rs10077427, and rs5744533), in order to determine the genotypes of 560 BC patients and 583 controls. The association of genotypes and BC was assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CIs) from logistic regression analyses. We found a statistically significant difference between patient and control groups in the POLK rs10077427 genotypic groups, excluding the recessive model. A positive correlation was also found between positive progesterone receptor (PR) status, higher Ki67 index, and rs10077427 polymorphism. For rs5744533 polymorphism, the codominant, dominant, and allele models frequencies were significantly higher in BC patients compared to healthy controls. Furthermore, our results indicated that rs5744533 SNP has a protective role in the postmenopausal women. However, we failed to find any associations between rs3213801 polymorphism and susceptibility to BC. Our results indicate that POLK polymorphisms may influence the risk of developing BC, and, because of this, may serve as a prognostic biomarker among Chinese women.

The low-fidelity DNA polymerases thought to be specialized in DNA damage processing are frequently misregulated in cancers. We show here that DNA polymerase kappa (polkappa), prone to replicate across oxidative and aromatic adducts and known to function in nucleotide excision repair (NER), is downregulated in colorectal tumour biopsies. Contrary to the replicative poldelta and polalpha, for which only activating domains were described, we identified an upstream 465-bp-long repressor region in the promoter of POLK. We also found an activating 237-bp region that includes stimulating protein-1 (SP1) and cyclic AMP-responsive element (CRE)-binding sites. Mutations at one CRE-binding site led to a dramatic 80% decrease in promoter activity. Alterations of the SP1-binding site also affected, to a lesser extent, the transcription. Gel shift assays confirmed the role played by CRE/SP1 recognition sequences. Moreover, ectopic expression of SP1 or CRE-binding protein (CREB) protein favoured polkappa transcription. Finally, we found that polkappa downexpression in colorectal biopsies correlated with a decreased level of CREB and SP1 transcripts. This work shows that the promoter of POLK is cis-controlled and suggests that silencing of CREB and SP1 proteins could contribute to downregulation of this repair polymerase in colorectal tumours.

DNA polymerase (pol) kappa is one of the so-called translesion polymerases involved in replication past DNA lesions. Bypass events have been studied with a number of chemical modifications with human pol kappa, and the conclusion has been presented, based on limited quantitative data, that the enzyme is ineffective at incorporating opposite DNA damage but proficient at extending beyond bases paired with the damage. Purified recombinant full-length human pol kappa was studied with a series of eight N(2)-guanyl adducts (in oligonucleotides) ranging in size from methyl- to -CH(2)(6-benzo[a]pyrenyl) (BP). Steady-state kinetic parameters (catalytic specificity, k(cat)/K(m)) were similar for insertion of dCTP opposite the lesions and for extension beyond the N(2)-adduct G:C pairs. Mispairing of dGTP and dTTP was similar and occurred with k(cat)/K(m) values approximately 10(-3) less than for dCTP with all adducts; a similar differential was found for extension beyond a paired adduct. Pre-steady-state kinetic analysis showed moderately rapid burst kinetics for dCTP incorporations, even opposite the bulky methyl(9-anthracenyl)- and BPG adducts (k(p) 5.9-10.3 s(-1)). The rapid bursts were abolished opposite BPG when alpha-thio-dCTP was used instead of dCTP, implying rate-limiting phosphodiester bond formation. Comparisons are made with similar studies done with human pols eta and iota; pol kappa is the most resistant to N(2)-bulk and the most quantitatively efficient of these in catalyzing dCTP incorporation opposite bulky guanine N(2)-adducts, particularly the largest (N(2)-BPG).

DNA polymerase kappa (POLkappa) is a low fidelity translesional DNA polymerase implicated in spontaneous and DNA damage-induced mutagenesis. We have previously shown that POLkappa was frequently overexpressed in human lung cancer tissues as compared with their matched non-tumorous tissue counterpart. In the present study, we found a close correlation between elevated POLkappa expression and p53 inactivation in lung cancer tissues. To investigate whether POLK expression might be regulated by p53, we have determined the transcriptional initiation site of POLK gene and examined its promoter activity in A549, H358-129, and PC-3 human lung cancer cell lines. Wild-type p53, but not a mutant p53 (R273H) devoid of the DNA-binding activity, strongly inhibited POLK promoter activity in these cells. In addition, POLK promoter exhibited a significantly higher activity in p53-/- murine embryo fibroblasts (MEF) than in p53+/- and p53+/+ MEF. These results link p53 status with POLkappa expression and suggest that loss of p53 function may in part contribute to the observed POLkappa upregulation in human lung cancers.

Prostate cancer is one of the most prevalent malignancies affecting men worldwide, and is the most frequent cancer among men in the USA, with an estimated 238,590 new cases and 29720 deaths in 2013 (NCI). DNA damage found in prostate cancer promotes tumor progression. Repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms. During replication, DNA polymerases frequently encounter unrepaired DNA lesions and posses 3′→5′ exonuclease proofreading activities. Such polymerases tend to stall at sites of damage, which can lead to replication fork collapse and which, unless rescued, can eventually cause large-scale genomic rearrangements and cell death. As a preventative measure, most organisms have alternative polymerases that are capable of ‘damage tolerance’ and can synthesize past DNA lesions by a mechanism called translesion synthesis (TLS). Polymerase kappa (Polκ) a Y-family polymerase participates in the process of TLS. Current study was designed to investigate the TLS of somatic missense mutations of Polκ such as p.F155S, p.L442F, p.G154E, p.E29K, p.E449K and p.E430G found after sequence analysis of prostate tumor DNA from 30 prostate cancer patients. We studied these variants to identify the nucleotide incorporated opposite to the apurinic (AP) site template, in vitro using site-specific tetrahydrofuran (AP site analog). We report that missense somatic mutations of Polκ at p.F155S, p.L442F, p.G154E, p.E29K, p.E449K mutants significantly decreases catalytic efficiency (k(cat)/Km). The biological consequence of an unrepaired DNA lesion is determined by whether it is bypassed or not, bypassed error prone. Our results provide evidence that mutations in the polymerase kappa gene can causes interference in Translesion DNA synthesis, this interference in pol κ function may play a role in prostate tumor progression. We acknowledge to Prof. Dr. Guengerich, for providing Polκ construct. This work was supported by DOD grant PC094628, and NIH grant 8 P20 GM103518. Citation Format: Santosh Yadav, Sudurika Mukhopadhyay, Nick Makridakis. Translesion DNA synthesis by DNA polymerase kappa somatic variants found in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2383. doi:10.1158/1538-7445.AM2014-2383

Background and Purpose: The therapeutic effect of many anticancer agents is due to induction of replication-blocking DNA lesions. Human DNA polymerase kappa (pol κ) is a trans-lesion synthesis polymerase that can catalyze direct replication past minor groove lesions, such as the one induced by mitomycin C. Pol κ has been found to be upregulated in ∼70% of gliomas and its upregulation associated with poorer prognosis in glioma patients. Therefore, small molecule inhibitors of pol κ in combination with chemotherapy may be useful in the treatment of cancer with upregulated pol κ expression. Methodology: Lead hits were initially identified from a screen of a library of ∼400,000 compounds at 5 different concentrations, using a strand-displacement DNA synthesis approach. From several thousand potential hits, these were systematically triaged, with 335 potential inhibitors of pol κ identified for further analyses. A direct, gel-based primer extension assay was used to test for inhibitory effects of these 335 bioactive compounds. In this assay, pol κ was preincubated with individual compounds, and added to 32P-labeled DNA substrate to initiate primer extensions. The reactions were incubated for 30 minutes at room temperature and terminated by the addition of a DNA-denaturing solution. The products were then separated through a 15% acrylamide gel and visualized with a PhosphorImager screen. Compounds that significantly inhibited the polymerization activity of pol κ were further tested for the ability to intercalate into DNA by incubation with double-stranded DNA for 15 minutes at room temperature, followed by separation through a 1% agarose gel. DNA was stained with ethidium bromide and visualized by Alpha Innotech ChemiImager. Results: Through a series of rigorous triaging procedures, this investigation identified a total of 42 small molecule inhibitors of pol κ with the estimated potencies being the low micromolar range or less. All 42 compounds were confirmed not to exhibit their inhibitory effects through DNA intercalation. These compounds need to be further tested with other members of the Y family polymerase to ensure their specificity to pol κ and determine if they are amenable to medicinal chemistry. Conclusion: Further investigation and characterization of these compounds will allow for a more efficient therapeutic approach in patients who have upregulated pol κ receiving chemotherapy. Citation Format: Christopher B. Sullivan, Irina G. Minko, Kinrin Yamanaka, Amanda K. McCullough, Stephen Lloyd. Identification of the potential inhibitors of DNA polymerase kappa. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3235. doi:10.1158/1538-7445.AM2014-3235

How do cells ensure that sister chromatids produced during DNA replication stay connected with each other until their separation in anaphase? New insight is provided by the discovery of DNA polymerase κ, which has been found to be required for building the connections between sister chromatids.

The Escherichia coli dinB gene encodes DNA polymerase (pol) IV, a protein involved in increasing spontaneous mutations in vivo. The protein-coding region of DINB1, the human ortholog of DNA pol IV, was fused to glutathione S-transferase and expressed in insect cells. The purified fusion protein was shown to be a template-directed DNA polymerase that we propose to designate pol kappa. Human pol kappa lacks detectable 3' --5' proofreading exonuclease activity and is not stimulated by recombinant human proliferating cell nuclear antigen in vitro. Between pH 6.5 and 8.5, human pol kappa possesses optimal activity at 37 degrees C over the pH range 6.5-7.5, and is insensitive to inhibition by aphidicolin, dideoxynucleotides, or NaCl up to 50 mm. Either Mg(2+) or Mn(2+) can satisfy a metal cofactor requirement for pol kappa activity, with Mg(2+) being preferred. Human pol kappa is unable to bypass a cisplatin adduct in the template. However, pol kappa shows limited bypass of an 2-acetylaminofluorene lesion and can incorporate dCTP or dTTP across from this lesion, suggesting that the bypass is potentially mutagenic. These results are consistent with a model in which pol kappa acts as a specialized DNA polymerase whose possible role is to facilitate the replication of templates containing abnormal bases, or possessing structurally aberrant replication forks that inhibit normal DNA synthesis.

Mammalian DNA polymerase kappa (pol kappa), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol kappa copies undamaged DNA with average single-base substitution and deletion error rates of 7 x 10(-3) and 2 x 10(-3), respectively. These error rates are high when compared to those of most other DNA polymerases. pol kappa also has unusual error specificity, producing a high proportion of T.CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol kappa can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol kappa is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.