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

• Published in: Molecular cell Vol. 81; no. 3; pp. 426 - 441.e8
• Main Authors: Jones, Mathew J.K., Gelot, Camille, Munk, Stephanie, Koren, Amnon, Kawasoe, Yoshitaka, George, Kelly A., Santos, Ruth E., Olsen, Jesper V., McCarroll, Steven A., Frattini, Mark G., Takahashi, Tatsuro S., Jallepalli, Prasad V.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.02.2021
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Animals; Ataxia Telangiectasia Mutated Proteins - genetics; Ataxia Telangiectasia Mutated Proteins - metabolism; ATR; Cdc7; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Checkpoint Kinase 1 - genetics; Checkpoint Kinase 1 - metabolism; chemical genetics; DDK; DNA replication; DNA Replication - drug effects; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Female; HCT116 Cells; HEK293 Cells; HeLa Cells; Humans; MERIT40; PDS5B; phosphoproteomics; 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; phosphoproteomics; DNA replication; MERIT40; chemical genetics; DDK; Cdc7; PDS5B; ATR
• ISSN: 1097-2765; 1097-4164; 1097-4164
• DOI: 10.1016/j.molcel.2021.01.004

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. [Display omitted] •DDKi causes spontaneous fork stalling and inhibits late origin firing•ATRi and Chk1i rescue origin firing in DDKi cells and frog egg extracts•DDKi blocks RPA loading, nascent-strand protection, and restart at stalled forks•MERIT40 and PDS5B are key DDK substrates in nascent-strand protection and restart Eukaryote genomes duplicate via spatially and temporally regulated firing of replication origins. Using chemical genetics and phosphoproteomics, Jones et al. show that human DDK promotes origin firing by counteracting the S phase checkpoint and enables the uncoupling, protection, and restart of stalled forks.