Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress |
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Authors: | Shimura Tsutomu Torres Michael J Martin Melvenia M Rao V Ashutosh Pommier Yves Katsura Mari Miyagawa Kiyoshi Aladjem Mirit I |
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Institution: | 1 Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, NIH, Bethesda, MD 20892-4255, USA 2 Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Sendai, Japan 3 Section of Radiation Biology, Graduate School of Medicine, The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan |
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Abstract: | 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 α 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. |
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Keywords: | APH aphidicolin ATM ataxia telangiectasia mutated ATR ATM and Rad3-related ATRkd ATR kinase dead BLM Bloom BS Bloom's syndrome BSA bovine serum albumin BrdU bromodeoxyuridine CldU 5-chloro-2&prime -deoxyuridine DMEM Dulbecco's modified Eagle's medium DNA-PK DNA-dependent protein kinase DNA-PKcs catalytic subunit of DNA-PK DSBs double-strand breaks FACS fluorescence-activated cell sorting FdU fluorodeoxyuridine HU hydroxyurea IdU 5-Iodo-2&prime -deoxyuridine MEM Minimum Essential Alpha Medium NHEJ non-homologous end-joining PBS phosphate-buffered saline pol α polymerase alpha PCNA proliferating cell nuclear antigen RPA replication protein A ssDNA single-strand DNA WT wild-type |
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