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

© 2001 Oxford University Press Nucleic Acids Research, 2001, Vol. 29, No. 6 1389–1398 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 Received October 3, 2000; Revised and Accepted January 8, 2001 ABSTRACT 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–singlestranded (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 nonWatson–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. INTRODUCTION The essential steps in homologous recombination are homologous pairing of two DNA chromosomes and exchange of DNA between them. Most of our knowledge about homologous recombination comes from studies of this process in Escherichia coli. The recombination events begin with a linear duplex DNA generated by double-strand breakage (or conjugation) that is either unwound by a helicase or degraded by a nuclease to produce a single-stranded (ss)DNA tail. RecA then polymerizes onto the ssDNA region to form a helical nucleoprotein filament, the RecA–ssDNA filament then searches for its homologous duplex DNA target. Once homology has been found, subsequent exchange of the individual DNA strands leads to heteroduplex DNA products (reviewed in 1–8). RecA, a 38 kDa polypeptide, performs a critical role in homologous recombination in E.coli. RecA binds to ssDNA to form a nucleoprotein filament that has been studied by 3D image reconstruction and by X-ray crystallography (9–11). The nucleoprotein filaments have a pitch of 95 Å with six RecA monomers per helical turn (9,10). A eukaryotic protein homologous to RecA, ScRAD51, has been shown to be essential in recombination and DNA damage repair in Saccharomyces cerevisiae (12). The human counterpart is termed human Rad51 (hRad51). Electron microscopy shows that both the yeast and human proteins form nucleoprotein filaments similar to those formed by RecA and in vitro experiments show that both proteins promote strand exchange (12–14). Thus, evidence suggests that the overall mechanism of recombination involving these strand exchange proteins has been conserved. The E.coli RecA system provides a valuable paradigm for the study of the mechanisms of homology searching and strand exchange. ssDNA coated with RecA searching for its homologous sequence buried in a non-RecA-coated duplex DNA initiates the homologous recombination process. Many biochemical methods (electron microscopy, filter binding, chemical probing and gel electrophoresis) have been used to study the intermediates and products of the strand exchange reaction promoted by RecA (reviewed in 1–8). However, it is still not clear how the RecA–ssDNA filament searches for and recognizes its homologous sequence in a duplex DNA to form a synaptic complex. There are two extreme models for initial recognition of homology. In the first model homologous DNA within the target duplex is locally unwound so that ssDNA in the RecA nucleoprotein filament can test for homology by forming Watson–Crick base pairs with the unwound segments (15–20). Homology searching would therefore involve multiple random encounters of the invading ssDNA–RecA filament with the duplex, followed by local melting of the duplex DNA and pairing with the invading ssDNA at each encounter site. In principle a second model is possible, with an intermediate in which the bases within the duplex target and ssDNA are held in registry; this alignment could be produced by non-Watson–Crick hydrogen bonds between the ssDNAs and double-stranded (ds)DNAs within the RecA filament (17,19,21). Thus, recognition of homology would occur via these non-Watson–Crick hydrogen bonds and the two strands of the target duplex DNA *To whom correspondence should be addressed. Tel: +1 302 831 1035; Fax: +1 302 831 6335; Email: Present address Ulf Marquardt, Raum K 205/206, Max-Planck-Institut für Biochemie, D-82152 Martinsried bei Muenchen, Germany 1390 Nucleic Acids Research, 2001, Vol. 29, No. 6 would remain paired by conventional Watson–Crick hydrogen bonds. This searching method might avoid the kinetic barriers associated with repeatedly breaking Watson–Crick hydrogen bonds in non-homologous duplex DNA regions before the homologous target is reached (22–25). In this paper we perform experiments aimed at deducing the influences of topology and the polarity of the RecA–ssDNA in formation of the synaptic complex (homologously paired joint molecule promoted by RecA), which might shed light on distinguishing between the two models described above. We have used a topological method and a restriction enzyme protection method with various linear or circular ssDNAs that were designed to limit the topological freedom to interwind with their target duplex DNA substrates in a RecA-mediated homologous pairing reaction. We demonstrate that, unlike its linear counterpart, a RecA-coated 520mer circular ssDNA can neither unwind nor form a stable joint molecule with a target circular 3 kb duplex DNA having a 120 bp region of homology with the ssDNA. We also show that supercoiling of the target duplex DNA enhances the efficiency of homologous pairing promoted by a linear, but not circular, RecA–ssDNA filament (with 120 nt homologous to its target duplex DNA) and that the polarity of the invading linear RecA–ssDNA has no effect on the efficiency of interwinding. The results suggest that topological freedom plays an important role in formation of stable synaptic complexes and that the RecA-mediated homology searching process is very likely coupled to unwinding of the dsDNA. Therefore, a triplex structure formed at the homologous region by non-Watson–Crick hydrogen (...truncated)