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Nucleic Acids Research Advance Access published online on May 12, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn261
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

LINE-1 methylation status of endogenous DNA double-strand breaks

Wichai Pornthanakasem1, Narisorn Kongruttanachok2, Chutipa Phuangphairoj1, Chotika Suyarnsestakorn2,3, Taweap Sanghangthum4, Sornjarod Oonsiri4, Wanpen Ponyeam1, Thatchawan Thanasupawat1, Oranart Matangkasombut5 and Apiwat Mutirangura1,*

1Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, 2Inter-Department Program of Biomedical Sciences, Faculty of Graduate School, Chulalongkorn University, Bangkok 10330, 3The National Center for Genetic Engineering and Biotechnology, Pathumthani 12120, 4Department of Radiology, Division of Radiation Oncology, Faculty of Medicine and 5Department of Microbiology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand

*To whom correspondence should be addressed. Tel: +662 256 4532; Fax: +662 254 1931; Email: apiwat.mutirangura{at}gmail.com

Received March 18, 2008. Revised April 17, 2008. Accepted April 17, 2008.

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.


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