DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells |
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| Authors: | Daniel Vare Petra Groth Rickard Carlsson Fredrik Johansson Klaus Erixon Dag Jenssen |
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| Institution: | 1. Faculty of Information Technology, Multimedia University, Cyberjaya, Malaysia;2. Faculty of Creative Industries, Universiti Tunku Abdul Rahman, Malaysia;3. Faculty of Information Science and Technology, Multimedia University, Melaka, Malaysia;3. Department of Chemistry, Wayne State University, Detroit, Michigan 48202;4. Center for Molecular Medicine and Genetics, Wayne State University, Wayne State University School of Medicine, Detroit, Michigan 48201;6. Department of Biochemistry, Microbiology and Immunology, Wayne State University School of Medicine, Detroit, Michigan 48201;5. Department of Pathology and Cancer Center, Massachusetts General Hospital, Boston, Massachusetts 02114;1. Research Institute of Advanced Materials (RIAM), Department of Materials Science and Engineering, Seoul National University, 56-1, Shinlim-dong, Gwanak-gu, Seoul 151-744, Republic of Korea;2. Korean Intellectual Property Office, Room 905, 4-Dong, Government Complex–Daejeon, Daejeon 302-701, Republic of Korea;3. Nano-Mechanical Systems Research Center, Intelligent and Precision Machinery Research Division, Korea Institute of Machinery and Materials, 104 Sinseongno, Yuseong-gu, Daejeon 305-343, Republic of Korea;4. Department of Fiber System Engineering, Dankook University, 126 Jukjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701, Republic of Korea |
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| Abstract: | DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells. |
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