Orderly progression through S-phase requires dynamic ubiquitylation and deubiquitylation of PCNA.

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.