PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast.

Yasukazu Daigaku,Tomoo Ogi,Thomas J Etheridge,Adam T Watson,Mayumi Nakayama,Antony M Carr,Mark A Osborne,Yuka Nakazawa,Izumi Miyabe,Lorraine S Symington

doi:10.1371/journal.pgen.1006789

Yasukazu Daigaku, Tomoo Ogi + Show 8 more

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https://doi.org/10.1371/journal.pgen.1006789

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Abstract

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling.

Highlights

It is well established that the replication machinery encounters a variety of obstacles and is designed with a degree of flexibility
In fission yeast PCNA is ubiquitylated during unperturbed S phase [18] and is not significantly further induced by UV-induced DNA damage during S-phase (Fig 1A and S1A and S1B Fig)
Cells arrested in early S phase by hydroxyurea maintained high levels of PCNA ubiquitylation (S1C Fig) and after irradiation in S phase PCNA ubiquitylation persisted for longer (Fig 1A), likely due to the slowed S phase progression

Summary

Introduction

It is well established that the replication machinery encounters a variety of obstacles and is designed with a degree of flexibility. This plasticity of DNA replication depends on both alternative components and regulation by post-translational modification. PCNA ubiquitylation on lysine164 regulates DNA damage tolerance (DTT). When replication is blocked by damaged DNA bases the Rad6-Rad E2-E3 ligase complex binds to single stranded DNA coated with RPA and mono-ubiquitylates PCNA to promote translesion DNA synthesises by non-canonical polymerases [10, 11]. Subsequent to mono-ubiquitylation, PCNA can be poly-ubiquitylated by the Ubc13-Mms2-Rad complex [12, 13] to initiate damage bypass by HR-dependent template switching [14]. The level and duration of PCNA ubiquitylation is regulated by constitutive deubiquitylation [15,16,17]

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