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Originally published In Press as doi:10.1074/jbc.M206962200 on July 29, 2002

J. Biol. Chem., Vol. 277, Issue 41, 38945-38953, October 11, 2002
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Structure of Reaction Intermediates Formed during Saccharomyces cerevisiae Rad51-catalyzed Strand Transfer*

Victor F. HolmesDagger , Francesca Scandellari§, Kirsten R. Benjamin, and Nicholas R. Cozzarelli||

From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720

The process by which the Saccharomyces cerevisiae strand transfer protein, Rad51, seeks out homologous sequences in vivo can be modeled by an in vitro reaction between a single-stranded DNA circle and a double-stranded linear DNA. In addition to the substrates and products, electrophoresis of reaction mixtures resolves two groups of low mobility bands. Here we show that the low mobility bands formed during strand transfer by Rad51 (or Escherichia coli RecA) represent joint molecules (JM) between the two substrates. One group, which we name JM1, is an obligatory reaction intermediate in which the complementary strand from the duplex substrate has been partially transferred to the single-stranded circle. Our assignment is based on pulse-chase and restriction enzyme digestion experiments and verified by electron microscopy. The slower moving group of bands, designated JM2, is formed by an unexpected reaction between JM1 and a second double-stranded linear substrate. Strand transfer of the second duplex initiates noncanonically from the end where the complementary strand is recessed. Thus JM2 is formed by two strand transfer reactions with the same single-stranded circular substrate but with opposite polarities. Finally, we show that the multiple sharp bands in JM1 and JM2 are the result of substrate sequences that pause strand transfer.

* This work was supported by grants (to N. R. C.) from the General Medical Institute of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Howard Hughes Medical Institute predoctoral fellow.

§ Present address: Dipartimento di Scienze e Technologie Agroambientali, Università di Bologna, 40126 Bologna, Italy.

Present address: Dept. of Biochemistry and Biophysics, University of California, San Francisco, CA 94143.

|| To whom correspondence should be addressed: 401 Barker Hall, 3204, University of California, Berkeley, CA 94720. E-mail: ncozzare@socrates.berkeley.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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