Lysine 63-Polyubiquitination Guards against Translesion Synthesis–Induced Mutations

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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. Genome instability is associated with increased cancer risk, and thus considerable effort has been put into unraveling the mechanisms underlying genome surveillance. Guarding the integrity of DNA are a number of DNA-repair and cell cycle–control systems. Insight into how these pathways become activated is crucially important to the understanding of carcinogenesis and in the development of cancer treatments. This study concerns a distinct pathway that promotes the tolerance of DNA damage during its replication phase. Prior attempts to investigate this pathway in human cells have been difficult due to extensive redundancy in the genes that carry out this process. Previous knowledge from lower organisms suggested the requirement for enzymes capable of constructing a chain of ubiquitin molecules linked in a specific manner. The authors used a novel approach to disrupt the formation of these ubiquitin chains in human cells and found that this caused a significant increase in mutations after exposure to UV light. Several lines of evidence implicate a family of error-prone enzymes, called translesion synthesis polymerases, in the formation of these mutations. Furthermore, they provide evidence suggesting that proliferating cell nuclear antigen (PCNA), a protein found at sites of replication, is the relevant target of these chains in human cells. These findings indicate that polyubiquitination guards against environmental carcinogenesis and contributes to genomic stability.

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