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Mol Cell. 2017 Oct 19;68(2):446-455.e3. doi: 10.1016/j.molcel.2017.09.018. Epub 2017 Oct 12.

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress.

Author information

1
Institute for Cancer Genetics and Department of Pediatrics and Genetics and Development, Columbia University, New York, NY 10032.
2
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USA.
3
Molecular Biology Program, Memorial Sloan Kettering Cancer Center (MSKCC), 1275 York Avenue, New York, NY 10065, USA; Weill-Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.
4
Department of Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden.
5
State Key Laboratory of Biotherapy and Cancer Center, Division of Abdominal Cancer, West China Hospital, Sichuan University, and National Collaborative Innovation Center for Biotherapy, Chengdu 610041, China.
6
Molecular Biology Program, Memorial Sloan Kettering Cancer Center (MSKCC), 1275 York Avenue, New York, NY 10065, USA.
7
Institute for Cancer Genetics and Department of Pediatrics and Genetics and Development, Columbia University, New York, NY 10032. Electronic address: zz2401@cumc.columbia.edu.

Abstract

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.

KEYWORDS:

ChIP-ssSeq; DNA replication checkpoint; Rad53; dNTP pools; eSPAN; fork collapse; lagging strand DNA synthesis; leading strand DNA synthesis

PMID:
29033319
PMCID:
PMC5802358
DOI:
10.1016/j.molcel.2017.09.018
[Indexed for MEDLINE]
Free PMC Article

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