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Nucleic Acids Research, 2001, Vol. 29, No. 6 1389-1398
© 2001 Oxford University Press

Topological testing of the mechanism of homology search promoted by RecA protein

Liping Cai, Ulf Marquardt, Zhaoqing Zhang, Mark J. Taisey and Junghuei Chen*

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA

To initiate homologous recombination, sequence similarity between two DNA molecules must be searched for and homology recognized. How the search for and recognition of homology occurs remains unproven. We have examined the influences of DNA topology and the polarity of RecA–single-stranded (ss)DNA filaments on the formation of synaptic complexes promoted by RecA. Using two complementary methods and various ssDNA and duplex DNA molecules as substrates, we demonstrate that topological constraints on a small circular RecA–ssDNA filament prevent it from interwinding with its duplex DNA target at the homologous region. We were unable to detect homologous pairing between a circular RecA–ssDNA filament and its relaxed or supercoiled circular duplex DNA targets. However, the formation of synaptic complexes between an invading linear RecA–ssDNA filament and covalently closed circular duplex DNAs is promoted by supercoiling of the duplex DNA. The results imply that a triplex structure formed by non-Watson–Crick hydrogen bonding is unlikely to be an intermediate in homology searching promoted by RecA. Rather, a model in which RecA-mediated homology searching requires unwinding of the duplex DNA coupled with local strand exchange is the likely mechanism. Furthermore, we show that polarity of the invading RecA–ssDNA does not affect its ability to pair and interwind with its circular target duplex DNA.

* To whom correspondence should be addressed. Tel: +1 302 831 1035; Fax: +1 302 831 6335; Email: junghuei{at}udel.edu Present address Ulf Marquardt, Raum K 205/206, Max-Planck-Institut für Biochemie, D-82152 Martinsried bei Muenchen, Germany


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