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

Mol Cell. 2021 Feb 4;81(3):426-441.e8. doi: 10.1016/j.molcel.2021.01.004.

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.

Keywords: ATR; Cdc7; DDK; DNA replication; MERIT40; PDS5B; chemical genetics; phosphoproteomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Checkpoint Kinase 1 / genetics
  • Checkpoint Kinase 1 / metabolism
  • DNA Replication* / drug effects
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Female
  • HCT116 Cells
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism
  • Replication Origin*
  • S Phase Cell Cycle Checkpoints
  • Substrate Specificity
  • Time Factors
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Xenopus laevis

Substances

  • Adaptor Proteins, Signal Transducing
  • BABAM1 protein, human
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • PDS5B protein, human
  • Protein Kinase Inhibitors
  • Transcription Factors
  • CDC7 protein, human
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • CHEK1 protein, human
  • Checkpoint Kinase 1
  • Protein Serine-Threonine Kinases