Abstract
DNA interstrand cross-links (ICLs) are the most cytotoxic lesions to eukaryotic genome and are repaired by both homologous recombination-dependent and -independent mechanisms. To better understand the role of lesion bypass polymerases in ICL repair, we investigated recombination-independent repair of ICLs in REV3 and REV1 deletion mutants constructed in avian DT40 cells and mouse embryonic fibroblast cells. Our results showed that Rev3 plays a major role in recombination-independent ICL repair, which may account for the extreme sensitivity of REV3 mutants to cross-linking agents. This result raised the possibility that the NER gap synthesis, when encountering an adducted base present in the ICL repair intermediate, can lead to recruitment of Rev3, analogous to the recruitment of polymerase eta during replicative synthesis. Indeed, the monoubiquitination-defective Proliferating Cell Nuclear Antigen (PCNA) mutant exhibits impaired recombination-independent ICL repair as well as drastically reduced mutation rate, indicating that the PCNA switch is utilized to enable lesion bypass during DNA repair synthesis. Analyses of a REV1 deletion mutant also revealed a significant reduction in recombination-independent ICL repair, suggesting that Rev1 cooperates with Rev3 in recombination-independent ICL repair. Moreover, deletion of REV3 or REV1 significantly altered the spectrum of mutations resulting from ICL repair, further confirming their involvement in mutagenic repair of ICLs.
Highlights
Bifunctional alkylating agents generate DNA interstrand cross-links (ICLs),2 which prevent strand separation required for essential DNA functions such as replication, transcription, and recombination
We show that Pol is likely the predominant lesion bypass polymerase in recombinationindependent ICL repair, with its involvement facilitated by the monoubiquitination of Proliferating Cell Nuclear Antigen (PCNA)
Examination of two Rev3ϩ/ϩ Mouse embryonic fibroblasts (MEFs) cell lines generated from a p53Ϫ/Ϫ background showed that cells transfected with psoralen-cross-linked pCMV-Luc substrate exhibited significant luciferase expression resulting from ICL removal (25.9 and 33.4% repair efficiency, respectively) (Fig. 1A)
Summary
REV3 and REV1 Play Major Roles in Recombination-independent Repair of DNA Interstrand Cross-links Mediated by Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA)*□S. Our investigations in mammalian cells have suggested an NER- and translesion synthesis-based errorprone mechanism of ICL repair in which the gap created by the NER dual incisions is resynthesized through participation of lesion bypass DNA polymerases [3, 4]. This error-prone mechanism may account for the mutagenic impact of ICLs. A similar mechanism has been demonstrated in budding yeast, suggesting that recombination-independent ICL repair may be a highly conserved mechanism in eukaryotes [5, 6]. Deletion of REV1 resulted in a major defect in recombination-independent ICL repair and reduced mutation formation, suggesting that Rev and Rev acts as one functional module during ICL repair
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