Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication
Research output: Contribution to journal › Journal article › Research › peer-review
Documents
- Fulltext
Accepted author manuscript, 3.27 MB, PDF document
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 language | English |
---|---|
Journal | Molecular Cell |
Volume | 81 |
Issue number | 3 |
Pages (from-to) | 426-441.e8 |
Number of pages | 24 |
ISSN | 1097-2765 |
DOIs | |
Publication status | Published - 2021 |
- Adaptor Proteins, Signal Transducing/genetics, Animals, Ataxia Telangiectasia Mutated Proteins/genetics, Cell Cycle Proteins/genetics, Checkpoint Kinase 1/genetics, DNA Replication/drug effects, DNA-Binding Proteins/genetics, Female, HCT116 Cells, HEK293 Cells, HeLa Cells, Humans, Phosphorylation, Protein Kinase Inhibitors/pharmacology, Protein-Serine-Threonine Kinases/genetics, Replication Origin, S Phase Cell Cycle Checkpoints, Substrate Specificity, Time Factors, Transcription Factors/genetics, Xenopus laevis
Research areas
ID: 259631737