Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin

doi:10.1083/jcb.150.2.283

Published online 24 July 2000. doi:10.1083/jcb.150.2.283

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© The Rockefeller University Press, 0021-9525/2000/7/283/ $5.00
The Journal of Cell Biology, Volume 150, Number 2, July 24, 2000 283-292

Original Article

Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin

Satoshi Tashiro^a,b, Joachim Walter^a, Akira Shinohara^d, Nanao Kamada^c, and Thomas Cremer^a
^a Institut für Anthropologie und Humangenetik, Universität München, München 80333, Germany
^b Department of Pediatrics, Faculty of Medicine
^c Research Institute Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8551, Japan
^d Department of Radiation and Cellular Oncology, University of Chicago, Chicago, Illinois 60637

Correspondence to: Satoshi Tashiro, Department of Pediatrics, Faculty of Medicine, Hiroshima University, Kasumi 1-2-3, Minamiku, Hiroshima 734-8551, Japan. Tel:81-82-257-5212 Fax:81-82-257-5214 E-mail:stashiro{at}mcai.med.hiroshima-u.ac.jp.

Rad51, a eukaryotic RecA homologue, plays a central role inhomologous recombinational repair of DNA double-strand breaks(DSBs) in yeast and is conserved from yeast to human. Rad51shows punctuate nuclear localization in human cells, calledRad51 foci, typically during the S phase (Tashiro, S., N. Kotomura,A. Shinohara, K. Tanaka, K. Ueda, and N. Kamada. 1996. Oncogene.12:2165–2170). However, the topological relationshipsthat exist in human S phase nuclei between Rad51 foci and damagedchromatin have not been studied thus far. Here, we report onultraviolet microirradiation experiments of small nuclear areasand on whole cell ultraviolet C (UVC) irradiation experimentsperformed with a human fibroblast cell line. Before UV irradiation,nuclear DNA was sensitized by the incorporation of halogenatedthymidine analogues. These experiments demonstrate the redistributionof Rad51 to the selectively damaged, labeled chromatin. Rad51recruitment takes place from Rad51 foci scattered throughoutthe nucleus of nonirradiated cells in S phase. We also demonstratethe preferential association of Rad51 foci with postreplicativechromatin in contrast to replicating chromatin using a doublelabeling procedure with halogenated thymidine analogues. Thisfinding supports a role of Rad51 in recombinational repair processesof DNA damage present in postreplicative chromatin.

Key Words: Rad51, DNA damage, microirradiation, postreplicative DNA repair,indirect immunofluorescence

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