The DNA binding properties of Saccharomyces cerevisiae Rad51 protein

Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-d ependent DNA strand exchange proteins. To explain some of the unique charac teristics of DNA strand exchange promoted by Rad51 protein, when compared w ith its prokaryotic homologue the Escherichia coil RecA protein, we analyze d the DNA binding properties of the Rad51 protein. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding s toichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphospha te) (ATP gamma S) can substitute for ATP, but binding in the presence of AT P gamma S requires more than a 5-fold stoichiometric excess of protein. Wit hout nucleotide co-factor, Rad51 protein binds both ssDNA and dsDNA but onl y at pH values lower than 6.8; in this case, the apparent binding stoichiom etry covers the range of 1 protein monomer per 6-9 nucleotides or base pair s. Therefore, Rad51 protein displays two distinct modes of DNA binding. The se binding modes are not inter-convertible; however, their initial selectio n is governed by ATP binding, On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding a bility, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during pre-synapsis, Rad51 protein int eracts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 p rotein inactivates the homologous dsDNA partner by directly binding to it.