Sequential replication-coupled destruction at G1/S ensures genome stability

Kate E. Coleman, Gavin D. Grant, Rachel A. Haggerty, Kristen Brantley, Etsuko Shibata, Benjamin D. Workman, Anindya Dutta, Dileep Varma, Jeremy E. Purvis, Jeanette Gowen Cook*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Timely ubiquitin-mediated protein degradation is fundamental to cell cycle control, but the precise degradation order at each cell cycle phase transition is still unclear. We investigated the degradation order among substrates of a single human E3 ubiquitin ligase, CRL4Cdt2, which mediates the S-phase degradation of key cell cycle proteins, including Cdt1, PR-Set7, and p21. Our analysis of synchronized cells and asynchronously proliferating live single cells revealed a consistent order of replication-coupled destruction during both S-phase entry and DNA repair; Cdt1 is destroyed first, whereas p21 destruction is always substantially later than that of Cdt1. These differences are attributable to the CRL4Cdt2 targeting motif known as the PIP degron, which binds DNA-loaded proliferating cell nuclear antigen (PCNADNA) and recruits CRL4Cdt2. Fusing Cdt1's PIP degron to p21 causes p21 to be destroyed nearly concurrently with Cdt1 rather than consecutively. This accelerated degradation conferred by the Cdt1 PIP degron is accompanied by more effective Cdt2 recruitment by Cdt1 even though p21 has higher affinity for PCNADNA. Importantly, cells with artificially accelerated p21 degradation display evidence of stalled replication in mid-S phase and sensitivity to replication arrest. We therefore propose that sequential degradation ensures orderly S-phase progression to avoid replication stress and genome instability.

Original languageEnglish (US)
Pages (from-to)1734-1746
Number of pages13
JournalGenes and Development
Volume29
Issue number16
DOIs
StatePublished - Aug 15 2015

Keywords

  • CDK
  • Cdt1
  • Replication stress
  • S phase
  • Ubiquitin
  • p21

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology

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