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BRCA1 promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade.

ABSTRACT: Breast cancer gene 1 (BRCA1) deficient cells not only are hypersensitive to double-strand breaks but also are hypersensitive to UV irradiation and other agents that cause replication blockade; however, the molecular mechanisms behind these latter sensitivities are largely unknown. Here, we report that BRCA1 promotes cell survival by directly regulating the DNA damage tolerance pathway in response to agents that create cross-links in DNA. We show that BRCA1 not only promotes efficient mono- and polyubiquitination of proliferating cell nuclear antigen (PCNA) by regulating the recruitment of replication protein A, Rad18, and helicase-like transcription factor to chromatin but also directly recruits translesion polymerases, such as Polymerase eta and Rev1, to the lesions through protein-protein interactions. Our data suggest that BRCA1 plays a critical role in promoting translesion DNA synthesis as well as DNA template switching.

SUBMITTER: Tian F

PROVIDER: S-EPMC3746927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

Project description:Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

| S-EPMC3892766 | biostudies-literature

Correction: The translesion DNA polymerases Pol ? and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase ?.

Project description:[This corrects the article DOI: 10.1371/journal.pgen.1007119.].

| S-EPMC5812558 | biostudies-literature

The translesion DNA polymerases Pol ? and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase ?.

Project description:Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase ? (Pol ?) subunits and/or between Pol ? and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol ? in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18?-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol ?. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ? TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol ? interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18?-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol ?-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol ? for PCNA access upon DNA damage.

| S-EPMC5760103 | biostudies-other

RFWD3 and translesion DNA polymerases contribute to PCNA modification-dependent DNA damage tolerance.

Project description:DNA damage tolerance pathways are regulated by proliferating cell nuclear antigen (PCNA) modifications at lysine 164. Translesion DNA synthesis by DNA polymerase η (Polη) is well studied, but less is known about Polη-independent mechanisms. Illudin S and its derivatives induce alkyl DNA adducts, which are repaired by transcription-coupled nucleotide excision repair (TC-NER). We demonstrate that in addition to TC-NER, PCNA modification at K164 plays an essential role in cellular resistance to these compounds by overcoming replication blockages, with no requirement for Polη. Polκ and RING finger and WD repeat domain 3 (RFWD3) contribute to tolerance, and are both dependent on PCNA modifications. Although RFWD3 is a FANC protein, we demonstrate that it plays a role in DNA damage tolerance independent of the FANC pathway. Finally, we demonstrate that RFWD3-mediated cellular survival after UV irradiation is dependent on PCNA modifications but is independent of Polη. Thus, RFWD3 contributes to PCNA modification-dependent DNA damage tolerance in addition to translesion DNA polymerases.

| S-EPMC9348633 | biostudies-literature

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.

Project description:Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.

| S-EPMC3169526 | biostudies-literature

Translesion DNA polymerases.

Project description:Living cells are continually exposed to DNA-damaging agents that threaten their genomic integrity. Although DNA repair processes rapidly target the damaged DNA for repair, some lesions nevertheless persist and block genome duplication by the cell's replicase. To avoid the deleterious consequence of a stalled replication fork, cells use specialized polymerases to traverse the damage. This process, termed "translesion DNA synthesis" (TLS), affords the cell additional time to repair the damage before the replicase returns to complete genome duplication. In many cases, this damage-tolerance mechanism is error-prone, and cell survival is often associated with an increased risk of mutagenesis and carcinogenesis. Despite being tightly regulated by a variety of transcriptional and posttranslational controls, the low-fidelity TLS polymerases also gain access to undamaged DNA where their inaccurate synthesis may actually be beneficial for genetic diversity and evolutionary fitness.

| S-EPMC3783050 | biostudies-literature

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Project description:Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.

| S-EPMC6914801 | biostudies-literature

Translesion-synthesis DNA polymerases participate in replication of the telomeres in Streptomyces.

Project description:Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5'-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3'-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ?dinB1 ?dinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.

| S-EPMC3273824 | biostudies-other

Translesion Polymerases Drive Microhomology-Mediated Break-Induced Replication Leading to Complex Chromosomal Rearrangements.

Project description:Complex genomic rearrangements (CGRs) are a hallmark of many human diseases. Recently, CGRs were suggested to result from microhomology-mediated break-induced replication (MMBIR), a replicative mechanism involving template switching at positions of microhomology. Currently, the cause of MMBIR and the proteins mediating this process remain unknown. Here, we demonstrate in yeast that a collapse of homology-driven break-induced replication (BIR) caused by defective repair DNA synthesis in the absence of Pif1 helicase leads to template switches involving 0-6 nt of homology, followed by resolution of recombination intermediates into chromosomal rearrangements. Importantly, we show that these microhomology-mediated template switches, indicative of MMBIR, are driven by translesion synthesis (TLS) polymerases Polζ and Rev1. Thus, an interruption of BIR involving fully homologous chromosomes in yeast triggers a switch to MMBIR catalyzed by TLS polymerases. Overall, our study provides important mechanistic insights into the initiation of MMBIR associated with genomic rearrangements, similar to those promoting diseases in humans.

| S-EPMC4688117 | biostudies-literature

Translesion DNA polymerases and cancer.

Project description:DNA repair has been regarded as an important barrier to carcinogenesis. The newly discovered field of translesion synthesis (TLS) has made it apparent that mammalian cells need distinct polymerases to efficiently and accurately bypass DNA lesions. Perturbation of TLS polymerase activity by mutation, loss of expression, etc. is expected to result in the accumulation of mutations in cells exposed to specific carcinogens. Furthermore, several TLS polymerases can modulate cellular sensitivity to chemotherapeutic agents. TLS genes and TLS gene variations may thus be attractive pharmacologic and/or pharmacogenetic targets. We review herein current data with regards to the potential contribution of the primary TLS polymerase genes to cancer, their interaction with pharmacologic agents, and identify areas of interest for further research.

| S-EPMC3434439 | biostudies-literature

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