Abstract
Eukaryotic cells possess several mechanisms to protect the integrity of their DNA against damage. These include cell-cycle checkpoints, DNA-repair pathways, and also a distinct DNA damage–tolerance system that allows recovery of replication forks blocked at sites of DNA damage. In both humans and yeast, lesion bypass and restart of DNA synthesis can occur through an error-prone pathway activated following mono-ubiquitination of proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, and recruitment of specialized translesion synthesis polymerases. In yeast, there is evidence for a second, error-free, pathway that requires modification of PCNA with non-proteolytic lysine 63-linked polyubiquitin (K63-polyUb) chains. Here we demonstrate that formation of K63-polyUb chains protects human cells against translesion synthesis–induced mutations by promoting recovery of blocked replication forks through an alternative error-free mechanism. Furthermore, we show that polyubiquitination of PCNA occurs in UV-irradiated human cells. Our findings indicate that K63-polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.
Highlights
In contrast to DNA-repair pathways, DNA damage tolerance (DDT) is characterized by bypass of DNA lesions rather than their direct removal or repair
UBC13/MMS2 is a unique Ub conjugase that synthesizes polyUb chains linked through K63–G76 bonds rather than through the typical K48–G76 bonds [13]
In order to directly investigate the functional importance of K63-linked polyUb chains in DDT, we employed a strategy similar to that first described in yeast, to inhibit assembly of these chains
Summary
In contrast to DNA-repair pathways, DNA damage tolerance (DDT) is characterized by bypass of DNA lesions rather than their direct removal or repair. Often referred to as RAD6dependent repair or post-replication repair, DDT requires interaction of the E2 ubiquitin (Ub) conjugase RAD6 and the E3 Ub ligase RAD18 at sites of DNA damage [4] They mediate mono-ubiquitination of proliferating cell nuclear antigen (PCNA) at K164 and subsequent recruitment of a specialized translesion synthesis (TLS) polymerase capable of replication past the lesion [5,6]. Saccharomyces cerevisiae RAD6 and RAD18 mutants that are unable to carry out DDT are highly sensitive to various genotoxic agents including UV irradiation and methyl methane sulfonate (MMS) [9] These mutants show a reduction in UV-induced mutations [10] that arises due to the inability to recruit the error-prone TLS polymerases [11]
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