Zebularine induces replication-dependent double-strand breaks which are preferentially repaired by homologous recombination |
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| Institution: | 1. Department of Cell Biology, University of Seville, Avda. Reina Mercedes 6, 41012 Seville, Spain,;2. Department of Pharmacology, Faculty of Pharmacy, University of Seville, c/Professor García González, No. 2, 41012, Seville, Spain;3. Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute, S-17121, Stockholm, Sweden;1. MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK;3. From the Departments of Molecular Genetics and The Ohio State University, Columbus, Ohio 43210;5. Human Nutrition and The Ohio State University, Columbus, Ohio 43210;4. Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210;6. the Department of Oncological Sciences and University of Utah School of Medicine, Salt Lake City, Utah 84132;12. the Microarray Core Facility and University of Utah School of Medicine, Salt Lake City, Utah 84112;8. Bioinformatics Shared Resource, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah 84112;3. From the CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, F-30207, France;;4. University of Warwick, Kirby Corner Road, Coventry, West Midlands, UK;1. Department of Pharmacology, Faculty of Medicine, University of Helsinki, Helsinki;2. Heart and Lung Center, Helsinki University Hospital, Helsinki;3. Department of Internal Medicine, Helsinki University Hospital, University of Helsinki, Helsinki;4. Department of Internal Medicine, South Karelia Central Hospital, Lappeenranta, Finland;1. Technological Institute of Food and Agriculture, Government of Extremadura (Spain), Hortofruenol Research Group, Avda. Adolfo Suárez, S/N, 06071 Badajoz, Spain;2. Food Colour and Quality Laboratory, Dept. Nutrition and Food Science, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain |
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| Abstract: | Zebularine is a second-generation, highly stable hydrophilic inhibitor of DNA methylation with oral bioavailability that preferentially target cancer cells. It acts primarily as a trap for DNA methyl transferases (DNMTs) protein by forming covalent complexes between DNMT protein and zebularine-substrate DNA. It’s well documented that replication-blocking DNA lesions can cause replication fork collapse and thereby to the formation of DNA double-strand breaks (DSB). DSB are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSB are non-homologous end joining (NHEJ) and homologous recombination (HR). Recently, multiple functions for the HR machinery have been identified at arrested forks. Here we investigate in more detail the importance of the lesions induced by zebularine in terms of DNA damage and cytotoxicity as well as the role of HR in the repair of these lesions. When we examined the contribution of NHEJ and HR in the repair of DSB induced by zebularine we found that these breaks were preferentially repaired by HR. Also we show that the production of DSB is dependent on active replication. To test this, we determined chromosome damage by zebularine while transiently inhibiting DNA synthesis. Here we report that cells deficient in single-strand break (SSB) repair are hypersensitive to zebularine. We have observed more DSB induced by zebularine in XRCC1 deficient cells, likely to be the result of conversion of SSB into toxic DSB when encountered by a replication fork. Furthermore we demonstrate that HR is required for the repair of these breaks. Overall, our data suggest that zebularine induces replication-dependent DSB which are preferentially repaired by HR. |
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| Keywords: | Zebularine Homologous recombination Replication DNA damage BRCA2 XRCC1 |
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