Role of TLS DNA polymerases eta and kappa in processing naturally occurring structured DNA in human cells.
ABSTRACT: Accurate DNA replication during S-phase is fundamental to maintain genome integrity. During this critical process, replication forks frequently encounter obstacles that impede their progression. While the regulatory pathways which act in response to exogenous replication stress are beginning to emerge, the mechanisms by which fork integrity is maintained at naturally occurring endogenous replication-impeding sequences remains obscure. Notably, little is known about how cells replicate through special chromosomal regions containing structured non-B DNA, for example, G4 quartets, known to hamper fork progression or trigger chromosomal rearrangements. Here, we have investigated the role in this process of the human translesion synthesis (TLS) DNA polymerases of the Y-family (pol eta, pol iota, and pol kappa), specialized enzymes known to synthesize DNA through DNA damage. We show that depletion by RNA interference of expression of the genes for Pol eta or Pol kappa, but not Pol iota, sensitizes U2OS cells treated with the G4-tetraplex interactive compound telomestatin and triggers double-strand breaks in HeLa cells harboring multiple copies of a G-rich sequence from the promoter region of the human c-MYC gene, chromosomally integrated as a transgene. Moreover, we found that downregulation of Pol kappa only raises the level of DSB in HeLa cells containing either one of two breakage hotspot structured DNA sequences in the chromosome, the major break region (Mbr) of BCL-2 gene and the GA rich region from the far right-hand end of the genome of the Kaposi Sarcoma associated Herpesvirus. These data suggest that naturally occurring DNA structures are physiological substrates of both pol eta and pol kappa. We discuss these data in the light of their downregulation in human cancers.
Project description:DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases η and κ. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation. Here, we investigated the consequences of mutant H-RAS G12V overexpression on the regulation of DNA polymerases in h-TERT immortalized and SV40-transformed human cells. Focusing on DNA polymerases associated with the replication fork, we demonstrate that DNA polymerases are depleted in a temporal manner in response to H-RAS G12V overexpression. The polymerases targeted for depletion, as cells display markers of senescence, include the Pol α catalytic subunit (POLA1), Pol δ catalytic and p68 subunits (POLD1 and POLD3), Pol η, and Pol κ. Both transcriptional and post-transcriptional mechanisms mediate this response. Pol η (POLH) depletion is sufficient to induce a senescence-like growth arrest in human foreskin fibroblast BJ5a cells, and is associated with decreased Pol α expression. Using an SV-40 transformed cell model, we observed cell cycle checkpoint signaling differences in cells with H-RasG12V-induced polymerase depletion, as compared to Pol η-deficient cells. Our findings contribute to our understanding of cellular events following oncogene activation and cellular transformation.
Project description:DNA polymerase (pol) iota, a member of the mammalian Y-family of DNA polymerases involved in translesion DNA synthesis, has been previously suggested to peculiarly utilize Hoogsteen base pairing for DNA synthesis opposite template purines, unlike pols eta and kappa, which utilize Watson-Crick (W-C) base pairing. To investigate the possible roles of Hoogsteen, W-C, and wobble base-pairing modes in the selection of nucleotides opposite template pyrimidines by human pol iota, we carried out kinetic analyses of incorporation of modified purine nucleoside triphosphates including 7-deazapurines, inosine, 2-aminopurine, 2,6-diaminopurine, and 6-chloropurine, which affect H-bonding in base-pair formation opposite template pyrimidines. Carbon substitution at the N7 atom of purine nucleoside triphosphates, which disrupts Hoogsteen base pairing, only slightly inhibited DNA synthesis opposite template pyrimidines by pol iota, which was not substantially different from human pols eta and kappa. Opposite template T, only the relative wobble stabilities (inferred from the potential numbers of H-bonding, steric, and electrostatic interactions but not measured) of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota but not the relative W-C or Hoogsteen stabilities, unlike pols eta and kappa. In contrast, opposite C, only the relative W-C stabilities of base pairs were positively correlated to the relative efficiencies of nucleotide incorporation by pol iota, as with pols eta and kappa. These results suggest that pol iota might not indispensably require Hoogsteen base pairing for DNA synthesis opposite pyrimidines but rather might prefer wobble base pairing in the selection of nucleotides opposite T and W-C base pairing opposite C.
Project description:Fanconi anemia (FA) is caused by defects in cellular responses to DNA crosslinking damage and replication stress. Given the constant occurrence of endogenous DNA damage and replication fork stress, it is unclear why complete deletion of FA genes does not have a major impact on cell proliferation and germ-line FA patients are able to progress through development well into their adulthood. To identify potential cellular mechanisms that compensate for the FA deficiency, we performed dropout screens in FA mutant cells with a whole genome guide RNA library. This uncovered a comprehensive genome-wide profile of FA pathway synthetic lethality, including <i>POLI</i> and <i>CDK4</i> As little is known of the cellular function of DNA polymerase iota (Pol ι), we focused on its role in the loss-of-function FA knockout mutants. Loss of both FA pathway function and Pol ι leads to synthetic defects in cell proliferation and cell survival, and an increase in DNA damage accumulation. Furthermore, FA-deficient cells depend on the function of Pol ι to resume replication upon replication fork stalling. Our results reveal a critical role for Pol ι in DNA repair and replication fork restart and suggest Pol ι as a target for therapeutic intervention in malignancies carrying an FA gene mutation.
Project description:DNA polymerase iota (pol iota) is a conserved Y family enzyme that is implicated in translesion DNA synthesis (TLS) but whose cellular functions remain uncertain. To test the hypothesis that pol iota performs TLS in cells, we compared UV-induced mutagenesis in primary fibroblasts derived from wild-type mice to mice lacking functional pol eta, pol iota, or both. A deficiency in mouse DNA polymerase eta (pol eta) enhanced UV-induced Hprt mutant frequencies. This enhanced UV-induced mutagenesis and UV-induced mutagenesis in wild-type cells were strongly diminished in cells deficient in pol iota, indicating that pol iota participates in the bypass of UV photoproducts in cells. Moreover, a clear strand bias among UV-induced base substitutions was observed in wild-type cells that was diminished in pol eta- and pol iota-deficient mouse cells and abolished in cells deficient in both enzymes. These data suggest that these enzymes bypass UV photoproducts in an asymmetric manner. To determine whether pol iota status affects cancer susceptibility, we compared the UV-induced skin cancer susceptibility of wild-type mice to mice lacking functional pol eta, pol iota, or both. Although pol iota deficiency alone had no effect, UV-induced skin tumors in pol eta-deficient mice developed 4 weeks earlier in mice concomitantly deficient in pol iota. Collectively, these data reveal functions for pol iota in bypassing UV photoproducts and in delaying the onset of UV-induced skin cancer.
Project description:All organisms have multiple DNA polymerases specialized for translesion DNA synthesis (TLS) on damaged DNA templates. Mammalian TLS DNA polymerases include Pol eta, Pol iota, Pol kappa, and Rev1 (all classified as "Y-family" members) and Pol zeta (a "B-family" member). Y-family DNA polymerases have highly structured catalytic domains; however, some of these proteins adopt different structures when bound to DNA (such as archaeal Dpo4 and human Pol kappa), while others maintain similar structures independently of DNA binding (such as archaeal Dbh and Saccharomyces cerevisiae Pol eta). DNA binding-induced structural conversions of TLS polymerases depend on flexible regions present within the catalytic domains. In contrast, noncatalytic regions of Y-family proteins, which contain multiple domains and motifs for interactions with other proteins, are predicted to be mostly unstructured, except for short regions corresponding to ubiquitin-binding domains. In this review we discuss how the organization of structured and unstructured regions in TLS polymerases is relevant to their regulation and function during lesion bypass.
Project description:Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene <i>POLH</i>) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.
Project description: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 (Pol?) could accumulate at laser-induced sites of damage in vivo resembling polymerases eta and iota. The recruitment was mediated through Pol? 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 Pol?-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 Pol? may have an important role in strand break repair following oxidative stress in vivo.
Project description:Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting translesion DNA synthesis (TLS) past sunlight-induced cyclobutane pyrimidine dimers. Despite its well-established role in TLS synthesis, the role of Pol eta in maintaining genome stability in the absence of external DNA damage has not been well explored. We show here that short hairpin RNA-mediated depletion of Pol eta from undamaged human cells affects cell cycle progression and the rate of cell proliferation and results in increased spontaneous chromosome breaks and common fragile site expression with the activation of ATM-mediated DNA damage checkpoint signaling. These phenotypes were also observed in association with modified replication factory dynamics during S phase. In contrast to that seen in Pol eta-depleted cells, none of these cellular or karyotypic defects were observed in cells depleted for Pol iota, the closest relative of Pol eta. Our results identify a new role for Pol eta in maintaining genomic stability during unperturbed S phase and challenge the idea that the sole functional role of Pol eta in human cells is in TLS DNA damage tolerance and/or repair pathways following exogenous DNA damage.
Project description:2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine in cooked foods, and is both mutagenic and carcinogenic. It has been suspected that the carcinogenicity of PhIP is derived from its ability to form DNA adducts, principally dG-C8-PhIP. To shed further light on the molecular mechanisms underlying the induction of mutations by PhIP, in vitro DNA synthesis analyses were carried out using a dG-C8-PhIP-modified oligonucleotide template. In this template, the dG-C8-PhIP adduct was introduced into the second G of the TCC GGG AAC sequence located in the 5' region. This represents one of the mutation hot spots in the rat Apc gene that is targeted by PhIP. Guanine deletions at this site in the Apc gene have been found to be preferentially induced by PhIP in rat colon tumors. DNA synthesis with A- or B-family DNA polymerases, such as Escherichia coli polymerase (pol) I and human pol delta, was completely blocked at the adducted guanine base. Translesional synthesis polymerases of the Y-family, pol eta, pol iota, pol kappa, and REV1, were also used for in vitro DNA synthesis analyses with the same templates. REV1, pol eta, and pol kappa were able to insert dCTP opposite dG-C8-PhIP, although the efficiencies for pol eta and pol kappa were low. pol kappa was also able to catalyze the extension reaction from the dC opposite dG-C8-PhIP, during which it often skipped over one dG of the triple dG sequence on the template. This slippage probably leads to the single dG base deletion in colon tumors.
Project description:DNA replication stress is often defined by the slowing or stalling of replication fork progression leading to local or global DNA synthesis inhibition. Failure to resolve replication stress in a timely manner contribute toward cell cycle defects, genome instability and human disease; however, the mechanism for fork recovery remains poorly defined. Here, we show that the translesion DNA polymerase (Pol) kappa, a DinB orthologue, has a unique role in both protecting and restarting stalled replication forks under conditions of nucleotide deprivation. Importantly, Pol kappa-mediated DNA synthesis during hydroxyurea (HU)-dependent fork restart is regulated by both the Fanconi Anemia (FA) pathway and PCNA polyubiquitination. Loss of Pol kappa prevents timely rescue of stalled replication forks, leading to replication-associated genomic instability, and a p53-dependent cell cycle defect. Taken together, our results identify a previously unanticipated role for Pol kappa in promoting DNA synthesis and replication stress recovery at sites of stalled forks.