Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication

Mathew J.K. Jones, Camille Gelot, Stephanie Munk, Amnon Koren, Yoshitaka Kawasoe, Kelly A. George, Ruth E. Santos, Jesper V. Olsen, Steven A. McCarroll, Mark G. Frattini, Tatsuro S. Takahashi, Prasad V. Jallepalli

Research output: Contribution to journalArticlepeer-review

Abstract

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.

Original languageEnglish
Pages (from-to)426-441.e8
JournalMolecular Cell
Volume81
Issue number3
DOIs
Publication statusPublished - Feb 4 2021

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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