TY - JOUR
T1 - Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage
AU - Yang, Yang
AU - Durando, Michael
AU - Smith-Roe, Stephanie L.
AU - Sproul, Chris
AU - Greenwalt, Alicia M.
AU - Kaufmann, William
AU - Oh, Sehyun
AU - Hendrickson, Eric A
AU - Vaziri, Cyrus
N1 - Funding Information:
Funding for open access charge: National Institutes of Health [ES09558 and ES016280 to C.V.].
PY - 2013/2
Y1 - 2013/2
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/gks1325
DO - 10.1093/nar/gks1325
M3 - Article
C2 - 23295675
AN - SCOPUS:84876349128
SN - 0305-1048
VL - 41
SP - 2296
EP - 2312
JO - Nucleic acids research
JF - Nucleic acids research
IS - 4
ER -