Abstract
Proliferating-cell nuclear antigen (PCNA) is a DNA sliding clamp with an essential function in DNA replication and a key role in tolerance to DNA damage by ensuring the bypass of lesions. In eukaryotes, DNA damage tolerance is regulated by ubiquitylation of lysine 164 of PCNA through a well-known control mechanism; however, the regulation of PCNA deubiquitylation remains poorly understood. Our work is a systematic and functional study on PCNA deubiquitylating enzymes (DUBs) in Schizosaccharomyces pombe. Our study reveals that the deubiquitylation of PCNA in fission yeast cells is a complex process that requires several ubiquitin proteases dedicated to the deubiquitylation of a specific subnuclear fraction of mono- and di-ubiquitylated PCNA or a particular type of poly-ubiquitylated PCNA and that there is little redundancy among these enzymes. To understand how DUB activity regulates the oscillatory pattern of ubiquitylated PCNA in fission yeast, we assembled multiple DUB mutants and found that a quadruple mutation of ubp2+, ubp12+, ubp15+, and ubp16+ leads to the stable accumulation of mono-, di-, and poly-ubiquitylated forms of PCNA, increases S-phase duration, and sensitizes cells to DNA damage. Our data suggest that the dynamic ubiquitylation and deubiquitylation of PCNA occurs during S-phase to ensure processive DNA replication.
Highlights
Living cells tolerate DNA damage during S-phase to prevent the risk of irreversible DNA replication fork collapse[1]
UbPCNA accumulation does not correlate with Usp[1] proteolysis when cells are exposed to other genotoxic agents, such as methyl methanesulfonate (MMS) and mitomycin C13,14, or the DNA replication blocking agent HU15, suggesting either that another human Proliferating-cell nuclear antigen (PCNA) deubiquitylating enzymes (DUBs) exists or that
We reasoned that in fission yeast, the ubiquitylation of PCNA might be a reversible process catalysed by deubiquitylating enzymes, as is the case in budding yeast
Summary
Living cells tolerate DNA damage during S-phase to prevent the risk of irreversible DNA replication fork collapse[1]. DNA damage tolerance is based on translesion synthesis (TLS), which involves a dual mechanism, primarily mediated by specialized low fidelity DNA polymerases called TLS-polymerases. These TLS polymerases can be mutagenic because they induce an error-prone process that causes mutations. The relevance of USP1 in reverting PCNA ubiquitylation when confronted with different DNA-damaging agents remains unclear. This is an important issue, because PCNA ubiquitylation is required for mammalian cell survival after UV irradiation and upon exposure to HU and MMS16,17. We propose that excessive DNA replication bypass interferes with the normal progression of DNA replication forks during S-phase
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