Branch migration during Rad51-promoted strand exchange proceeds in either direction

Abstract

The Saccharomyces cerevisiae Rad51 protein is important for genetic recombination and repair of DNA double-strand breaks in vivo and can promote strand exchange between linear double-stranded DNA and circular singlestranded DNA in vitro. However, unlike Escherichia coli RecA, Rad51 requires an overhanging complementary 3 or 5 end to initiate strand exchange; given that fact, we previously surmised that the fully exchanged molecules resulted from branch migration in either direction depending on which type of end initiated the joint molecule. Our present experiments confirm that branch migration proceeds in either direction, the polarity depending on whether a 3 or 5 end initiates the joint molecules. Furthermore, heteroduplex DNA is formed rapidly, first at the overhanging end of the linear double-stranded DNA's complementary strand and then more slowly by progressive lengthening of the heteroduplex region until strand exchange is complete. Although joint molecule formation occurs equally efficiently when initiated with a 3 or 5 overhanging end, branch migration proceeds more rapidly when it is initiated by an overhanging 3 end, i.e., in the 5 to 3 direction with respect to the single-stranded DNA.Saccharomyces cerevisiae Rad51, like Escherichia coli RecA, catalyzes joint molecule formation and complete strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) (1-3). Both require ssDNA binding protein and ATP to effect the strand exchange (1, 3, 4). However, unlike RecA, Rad51 requires an overhanging complementary 3Ј or 5Ј end on the dsDNA to initiate strand exchange; given that fact, we surmised (3) that the fully exchanged molecules resulted from branch migration in either direction depending on which type of end initiated the joint molecule. In that work, however, only joint and fully exchanged products were monitored, and the course and direction of branch migration were not measured directly.Our present experiments confirm that heteroduplex DNA is formed rapidly, first at the overhanging end of the linear dsDNA's complementary strand and then more slowly by progressive lengthening of the heteroduplex region until strand exchange is complete. Furthermore, branch migration proceeds in either direction, the polarity depending on whether a 3Ј or 5Ј end initiates the joint molecules. Although joint molecule formation occurs equally efficiently when initiated with a 3Ј or 5Ј overhanging end, branch migration proceeds more rapidly when it is initiated by an overhanging 3Ј end, i.e., in the 5Ј to 3Ј direction with respect to the ssDNA. MATERIALS AND METHODSProteins. Yeast Rad51 was expressed in insect cells and purified as described previously (3); yeast replication protein A (RPA) was expressed in E. coli by using the plasmid pJM126 (provided by S. Brill and B. Stillman, Cold Spring Harbor Laboratory, NY) and purified as described (5). The concentrations of Rad51 and RPA were determined by using extinction coefficients of 1.26 ϫ 10 3 and 8.8 ϫ 10 4 at 280 nm, respe...

Structure of Reaction Intermediates Formed duringSaccharomyces cerevisiae Rad51-catalyzed Strand Transfer

“…Although these species have been frequently observed for both RecA and Rad51 reactions (10,15,(17)(18)(19)(20)(21)(22), their structure and precise role in strand transfer have been unclear. Kinetic analysis of the yeast Rad51 reaction has shown that low mobility species accumulate in an early fast phase and then dissipate during a second, slower phase as products accumulate (16). In this report we show that the low mobility bands formed by yeast Rad51 represent joint molecules (JM) between the two substrates.…”

“…1B), implying that the pauses are inherent to the DNA sequence. Direct sequencing of the dsL and ssC substrates ruled out point mutations which are known to pause branch migration (16,21,39). Nor do pause sites correspond to obvious GC-rich sequences that could slow denaturation of the duplex.…”

“…This model takes advantage of the unique ability of Rad51 to promote strand exchange in both the 3Ј to 5Ј and 5Ј to 3Ј direction (13). However, whereas Namsaraev and Berg have reported that strand transfer initiates only from the complementary overhang (16), in this model the second dsL substrate must initiate via the end where the complementary strand is recessed. We refer to this model for JM2 formation as reverse initiation.…”

“…Namsaraev and Berg (16), however, reported that in vitro strand transfer requires that the duplex substrate have a single-stranded overhang that is complementary to the DNA in the filament. The reaction initiates at the complementary overhang, which can be a 5Ј or 3Ј end, and proceeds across the duplex substrate (16). Thus, the polarity of the Rad51 strand transfer reaction is determined only by where initiation occurs.…”

Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

“…33 However, it has been shown that the preferred substrate for Rad51-mediated homologous pairing is double-stranded (ds) DNA with ss DNA tails. 34, 35 Here we demonstrate that direct in vivo gene modification can be achieved using a novel approach based on branched oligonucleotides (b-oligonucleotides). The b-oligonucleotides that are presented in this article consist of ds DNA with 5 0 ss DNA tails coupled together by a novel baseless deoxyribose (dR) branching monomer.…”