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

Yang Yang, Michael Durando, Stephanie L. Smith-Roe, Chris Sproul, Alicia M. Greenwalt, William Kaufmann, Sehyun Oh, Eric A Hendrickson, Cyrus Vaziri

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Abstract

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 2O2-induced DNA damage. UVC and H2O2 treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G0, G1 and S-phase. Rad18 was important for repressing H2O2-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G1, indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H2O2-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 2O2-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G1. In contrast with G1-synchronized cultures, S-phase cells were H2O2-sensitive following Rad18-depletion. Weconclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polg is dispensable for damage-tolerance in G1 (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.

Original languageEnglish (US)
Pages (from-to)2296-2312
Number of pages17
JournalNucleic acids research
Volume41
Issue number4
DOIs
StatePublished - Feb 1 2013

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Yang, Y., Durando, M., Smith-Roe, S. L., Sproul, C., Greenwalt, A. M., Kaufmann, W., Oh, S., Hendrickson, E. A., & Vaziri, C. (2013). Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage. Nucleic acids research, 41(4), 2296-2312. https://doi.org/10.1093/nar/gks1325