Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans.
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ABSTRACT:
SUBMITTER: Sebesta M
PROVIDER: S-EPMC3744802 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Unrepaired DNA damage hinders the maintenance of genome integrity because it blocks the catalytic activity of replicase. The stalled replication fork can be processed through either translesion synthesis (TLS) with specific polymerases, or replication using the undamaged template. To investigate how TLS activities are regulated and how the stalled replication fork is processed in plants, reversion frequencies and homologous recombination (HR) frequencies were analyzed using GUS-based substrates. The HR frequencies in plants deficient in DNA polymerase ? (Pol ?) or Rev1 were higher than that in wildtype plants under normal conditions, and were significantly increased by ultraviolet light irradiation. Heat shock protein (HSP) 90 is known to be involved in various stress responses. To examine the role of HSP90 in the regulation of damage tolerance, we analyzed reversion frequencies and HR frequencies in plants grown in the presence of a HSP inhibitor, geldanamycin (GDA). Reversion frequency was lower in GDA-treated plants than in mock-treated plants. Though the HR frequency was higher in GDA-treated wildtype plants than in mock-treated plants, no significant difference was detected in Rev1-deficient plants. In yeast, TLS polymerases interacted with each other or with a replication clump component, proliferating cell nuclear antigen (PCNA). HSP90 interacted with REV1 or REV7 in Nicotiana benthamiana cells. These results suggest that HSP90 interacts with TLS polymerase(s), which promotes error-prone TLS in plants.
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Project description:In metazoans, the mechanism by which DNA is synthesized during homologous recombination repair of double-strand breaks is poorly understood. Specifically, the identities of the polymerase(s) that carry out repair synthesis and how they are recruited to repair sites are unclear. Here, we have investigated the roles of several different polymerases during homologous recombination repair in Drosophila melanogaster. Using a gap repair assay, we found that homologous recombination is impaired in Drosophila lacking DNA polymerase zeta and, to a lesser extent, polymerase eta. In addition, the Pol32 protein, part of the polymerase delta complex, is needed for repair requiring extensive synthesis. Loss of Rev1, which interacts with multiple translesion polymerases, results in increased synthesis during gap repair. Together, our findings support a model in which translesion polymerases and the polymerase delta complex compete during homologous recombination repair. In addition, they establish Rev1 as a crucial factor that regulates the extent of repair synthesis.
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Project description:Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) ? is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Pol? can function independently of PCNA modification and that Pol? can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Pol?, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Pol? and Pol? are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Pol? and establish Pol? as an important backup polymerase in the absence of Pol? in response to MMS-induced DNA damage.
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Project description:Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.
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Project description:Here we found that ILF3 prefers to bind telomere R-loops and protects telomere from aberrant homologous recombination. ILF3 knockout induces TERRA aggregation onto telomere and activates telomere DNA damage response (DDR). Furthermore, ILF3 deficiency disrupts telomere homeostasis and induces abnormal ALT-mediated telomere lengthening
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