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. Gan et al. find that Rad53 prevents fork collapse via coordinating the unwinding of double-stranded DNA by the replicative helicase with synthesis of both leading and lagging strand. They observed that long stretches of single-stranded leading-strand template coated with RPA are produced in rad53-1 mutant cells under replication stress.
Bibliographical noteFunding Information:
We thank Drs. Bruce Stillman, Xiaolan Zhao, Rodney Rothstein, Alberto Ciccia, Lorraine Symington, Jean Gautier, and Erik Johansson for discussions and comments on this manuscript. We thank Drs. Symington, Diffley, Wittenberg, Kunkel, and Kolodner for yeast strains and plasmids and Dr. Brill for antibodies against yeast RPA. This study is supported by NIH grants R35GM118015 (Z.Z.) and R01GM107239 (D.R.) and by the Swedish Cancer Society and the Swedish Research Council (A.C.).
© 2017 Elsevier Inc.
- DNA replication checkpoint
- dNTP pools
- fork collapse
- lagging strand DNA synthesis
- leading strand DNA synthesis