Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress.
Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase alpha from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.
Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-4255, USA.
Tsutomu
T
Shimura
Michael J
MJ
Torres
Melvenia M
MM
Martin
V Ashutosh
VA
Rao
Yves
Y
Pommier
Mari
M
Katsura
Kiyoshi
K
Miyagawa
Mirit I
MI
Aladjem
D016590:Aphidicolin / Q000494:pharmacology; D001816:Bloom Syndrome / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D020552:Comet Assay; D004247:DNA / Q000032:analysis; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004720:Endonucleases / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects; D019085:Fluorescent Antibody Technique, Direct; D006657:Histones / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism
D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D018809:Proliferating Cell Nuclear Antigen; D016590:Aphidicolin; D004247:DNA; D006861:Hydrogen Peroxide; D004720:Endonucleases; C498772:MUS81 protein, human; D004265:DNA Helicases