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DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis.

ABSTRACT: Deoxyribonucleic acid (DNA) lesions encountered during replication are often bypassed using DNA damage tolerance (DDT) pathways to avoid prolonged fork stalling and allow for completion of DNA replication. Rad18 is a central E3 ubiquitin ligase in DDT, which exists in a monoubiquitinated (Rad18•Ub) and nonubiquitinated form in human cells. We find that Rad18 is deubiquitinated when cells are treated with methyl methanesulfonate or hydrogen peroxide. The ubiquitinated form of Rad18 does not interact with SNF2 histone linker plant homeodomain RING helicase (SHPRH) or helicase-like transcription factor, two downstream E3 ligases needed to carry out error-free bypass of DNA lesions. Instead, it interacts preferentially with the zinc finger domain of another, nonubiquitinated Rad18 and may inhibit Rad18 function in trans. Ubiquitination also prevents Rad18 from localizing to sites of DNA damage, inducing proliferating cell nuclear antigen monoubiquitination, and suppressing mutagenesis. These data reveal a new role for monoubiquitination in controlling Rad18 function and suggest that damage-specific deubiquitination promotes a switch from Rad18•Ub-Rad18 complexes to the Rad18-SHPRH complexes necessary for error-free lesion bypass in cells.

SUBMITTER: Zeman MK

PROVIDER: S-EPMC4107794 | biostudies-literature |

REPOSITORIES: biostudies-literature

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DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis

Project description: Not available

| S-JCBD-201311063 | bioimages

Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage.

Project description:The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.

| S-EPMC3575850 | biostudies-literature

Regulation of Rad6/Rad18 Activity During DNA Damage Tolerance.

Project description:Replicative polymerases (pols) cannot accommodate damaged template bases, and these pols stall when such offenses are encountered during S phase. Rather than repairing the damaged base, replication past it may proceed via one of two DNA damage tolerance (DDT) pathways, allowing replicative DNA synthesis to resume. In translesion DNA synthesis (TLS), a specialized TLS pol is recruited to catalyze stable, yet often erroneous, nucleotide incorporation opposite damaged template bases. In template switching, the newly synthesized sister strand is used as a damage-free template to synthesize past the lesion. In eukaryotes, both pathways are regulated by the conjugation of ubiquitin to the PCNA sliding clamp by distinct E2/E3 pairs. Whereas monoubiquitination by Rad6/Rad18 mediates TLS, extension of this ubiquitin to a polyubiquitin chain by Ubc13-Mms2/Rad5 routes DDT to the template switching pathway. In this review, we focus on the monoubiquitination of PCNA by Rad6/Rad18 and summarize the current knowledge of how this process is regulated.

| S-EPMC5592839 | biostudies-literature

USP7-mediated deubiquitination differentially regulates CSB but not UVSSA upon UV radiation-induced DNA damage.

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RAD18 transmits DNA damage signalling to elicit homologous recombination repair.

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Meiotic functions of RAD18.

Project description:RAD18 is an ubiquitin ligase that is involved in replication damage bypass and DNA double-strand break (DSB) repair processes in mitotic cells. Here, we investigated the testicular phenotype of Rad18-knockdown mice to determine the function of RAD18 in meiosis, and in particular, in the repair of meiotic DSBs induced by the meiosis-specific topoisomerase-like enzyme SPO11. We found that RAD18 is recruited to a specific subfraction of persistent meiotic DSBs. In addition, RAD18 is recruited to the chromatin of the XY chromosome pair, which forms the transcriptionally silent XY body. At the XY body, RAD18 mediates the chromatin association of its interaction partners, the ubiquitin-conjugating enzymes HR6A and HR6B. Moreover, RAD18 was found to regulate the level of dimethylation of histone H3 at Lys4 and maintain meiotic sex chromosome inactivation, in a manner similar to that previously observed for HR6B. Finally, we show that RAD18 and HR6B have a role in the efficient repair of a small subset of meiotic DSBs.

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Dub3 controls DNA damage signalling by direct deubiquitination of H2AX.

Project description:A crucial event in the DNA damage response is the phosphorylation and subsequent ubiquitination of H2AX, required for the recruitment of proteins involved in DNA repair. Here we identify a novel regulator of this process, the ubiquitin hydrolase Dub3. Overexpression of wild type, but not catalytic inactive, Dub3 decreases the DNA damage-induced mono-ubiquitination of H2A(X) whereas downregulation of Dub3 has the opposite effect. Dub3 overexpression abrogates focus formation of 53BP1 and BRCA1 in response to genotoxic stress. However, focus formation of MDC1 and ?H2AX, earlier events in this response, are unaffected by Dub3 overexpression. We show that Dub3 counteracts H2AX E3 ligases RNF8 and RNF168. Moreover, Dub3 and H2AX interact and Dub3 deubiquitinates H2AX in vitro. Importantly, overexpression of Dub3 delays H2AX dephosphorylation and recovery of MDC1 focus formation at later time points after DNA damage, whereas H2AX dephosphorylation at later time points is faster after Dub3 depletion. Altogether these results show that Dub3 regulates a correct DNA damage response by controlling H2AX ubiquitination.

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Project description:Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs.

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SETD1A Methyltransferase Is Physically and Functionally Linked to the DNA Damage Repair Protein RAD18.

Project description:SETD1A is a SET domain-containing methyltransferase involved in epigenetic regulation of transcription. It is the main catalytic component of a multiprotein complex that methylates lysine 4 of histone H3, a histone mark associated with gene activation. In humans, six related protein complexes with partly nonredundant cellular functions share several protein subunits but are distinguished by unique catalytic SET-domain proteins. We surveyed physical interactions of the SETD1A-complex using endogenous immunoprecipitation followed by label-free quantitative proteomics on three subunits: SETD1A, RBBP5, and ASH2L. Surprisingly, SETD1A, but not RBBP5 or ASH2L, was found to interact with the DNA damage repair protein RAD18. Reciprocal RAD18 immunoprecipitation experiments confirmed the interaction with SETD1A, whereas size exclusion and protein network analysis suggested an interaction independent of the main SETD1A complex. We found evidence of SETD1A and RAD18 influence on mutual gene expression levels. Further, knockdown of the genes individually showed a DNA damage repair phenotype, whereas simultaneous knockdown resulted in an epistatic effect. This adds to a growing body of work linking epigenetic enzymes to processes involved in genome stability.

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Pyrosequencing: applicability for studying DNA damage-induced mutagenesis.

Project description:Site-specifically modified DNAs are routinely used in the study of DNA damage-induced mutagenesis. These analyses involve the creation of DNA vectors containing a lesion at a pre-determined position, DNA replication, and detection of mutations at the target site. The final step has previously required the isolation of individual DNA clones, hybridization with radioactively labeled probes, and verification of mutations by Sanger sequencing. In the search for an alternative procedure that would allow direct quantification of sequence variants in a mixed population of DNA molecules, we evaluated the applicability of pyrosequencing to site-specific mutagenesis assays. The progeny DNAs were analyzed that originated from replication of N(6) -(deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG)-containing vectors in primate cells, with the lesion being positioned in the 5'-GCNGG-3' sequence context. Pyrosequencing detected ?8% G to T transversions and ?3.5% G to A transitions, a result that was in excellent agreement with frequencies previously measured by the standard procedure (Earley LF et al. [2013]: Chem Res Toxicol 26:1108-1114). However, ?3.5% G to C transversions and ?2.0% deletions could not be detected by pyrosequencing. Consistent with these observations, the sensitivity of pyrosequencing for measuring the single deoxynucleotide variants differed depending on the deoxynucleotide identity, and in the given sequence contexts, was determined to be ?1-2% for A and T and ?5% for C. Pyrosequencing of other DNA isolates that were obtained following replication of MeFapy-dG-containing vectors in primate cells or Escherichia coli, identified several additional limitations. Collectively, our data demonstrated that pyrosequencing can be used for studying DNA damage-induced mutagenesis as an effective complementary experimental approach to current protocols.

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