TY - JOUR
T1 - Human DDK rescues stalled forks and counteracts checkpoint inhibition at unfired origins to complete DNA replication
AU - Jones, Mathew J.K.
AU - Gelot, Camille
AU - Munk, Stephanie
AU - Koren, Amnon
AU - Kawasoe, Yoshitaka
AU - George, Kelly A.
AU - Santos, Ruth E.
AU - Olsen, Jesper V.
AU - McCarroll, Steven A.
AU - Frattini, Mark G.
AU - Takahashi, Tatsuro S.
AU - Jallepalli, Prasad V.
N1 - Funding Information:
We thank Thomas Kelly for helpful discussions; Bruce Clurman, Gregory David, and Kevan Shokat for gifts of cell lines, plasmids, and chemicals; and George Church for sharing plasmids via Addgene. This work was supported by grants from the National Institutes of Health (R01GM094972, P30CA008748, and DP2GM123495), the Novo Nordisk Foundation ( NNF14CC0001 ), the Japan Society for the Promotion of Science (MEXT/JSPS KAKENHI JP17H01876 , JP20H03186 , and JP20H05392 ), the Harold G. and Leila Y. Mathers Charitable Foundation , the William H. and Alice Goodwin Center for Experimental Therapeutics , the Geoffrey Beene Cancer Research Center , and the Functional Genomics Initiative at MSKCC .
Publisher Copyright:
© 2021 Elsevier Inc.
PY - 2021/2/4
Y1 - 2021/2/4
N2 - 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.
AB - 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.
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U2 - 10.1016/j.molcel.2021.01.004
DO - 10.1016/j.molcel.2021.01.004
M3 - Article
C2 - 33545059
AN - SCOPUS:85100388860
SN - 1097-2765
VL - 81
SP - 426-441.e8
JO - Molecular Cell
JF - Molecular Cell
IS - 3
ER -