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bioRxiv · 10.1101/2024.12.16.628697

USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that enriches for monoubiquitinated PCNA

Keijzer, N.·Sakoltchik, J.·Majumder, K.·van Lil, N.·El Oualid, F.·Fish, A.·Sixma, T. K.
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Abstract

DNA damage tolerance (DDT) is an important pathway that allows our cells to bypass DNA lesions during replication. DDT is orchestrated by ubiquitination of PCNA: Monoubiquitination (PCNA-Ub) initiates recruitment of TLS polymerases but also serves as substrate for K63-linked polyubiquitination that leads to HR-mediated bypass mechanisms. Recent work on USP1/UAF1 inhibition revealed that K48-linked chains are also formed on PCNA, resulting in its proteasomal degradation. USP1/UAF1 is established as deubiquitinating enzyme (DUB) for PCNA-Ub, but little is known about deubiquitination of chains on PCNA. Here we show that USP1/UAF1 cleaves both K48 and K63-linked ubiquitin chains on PCNA efficiently, using an exo-cleavage mechanism. Kinetic analysis reveals that USP1/UAF1 prefers cleaving the ubiquitin-ubiquitin bond over cleavage of the ubiquitin-PCNA isopeptide bond and therefore treats poly- and monoubiquitinated PCNA as different substrates. A cryo-EM structure of USP1/UAF1 with a K63-diubiquitin and structure-based mutagenesis reveals that its mechanistic preference is maintained in evolution. Its kinetic mechanism results in relative enrichment of monoubiquitinated PCNA that could initially promote TLS over HR-like bypass. Taken together, these results suggest that USP1/UAF1 could be important in temporary protection of PCNA against K48- and K63-linked polyubiquitination and highlight this DUB as a potential regulator of DDT pathway choice.

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Keijzer, N., Sakoltchik, J., Majumder, K., van Lil, N., El Oualid, F., Fish, A., Sixma, T. K.. 2024-12-16. USP1/UAF1 targets polyubiquitinated PCNA with an exo-cleavage mechanism that enriches for monoubiquitinated PCNA. https://doi.org/10.1101/2024.12.16.628697

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