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Published online 24 July 2000. doi:10.1083/jcb.150.2.283
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© The Rockefeller University Press, 0021-9525/2000//283 $5.00
The Journal of Cell Biology, Volume 150, Number 2, , 2000 283-292

Original Article

Rad51 Accumulation at Sites of DNA Damage and in Postreplicative Chromatin



Satoshi Tashiroa,b, Joachim Waltera, Akira Shinoharad, Nanao Kamadac, and Thomas Cremera

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
Department of Pediatrics, Faculty of Medicine, Hiroshima University, Kasumi 1-2-3, Minamiku, Hiroshima 734-8551, Japan.81-82-257-521481-82-257-5212

stashiro{at}mcai.med.hiroshima-u.ac.jp

Rad51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 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 relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. Here, we report on ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. We also demonstrate the preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin.

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

© 2000 The Rockefeller University Press

Abbreviations used in this paper: BrdU, bromodeoxyuridine; CldU, chlorodeoxyuridine; DSBs, DNA double strand breaks; IdU, iododeoxyuridine; SSBs, DNA single strand breaks; ssDNA, single stranded DNA.


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