Rad51, the major eukaryotic homologous recombinase, is important for the repair of DNA damage and the maintenance of genomic diversity and stability. The active form of this DNA-dependent ATPase is a helical filament within which the search for homology and strand exchange occurs. Here we present the crystal structure of a Saccharomyces cerevisiae Rad51 filament formed by a gain-of-function mutant. This filament has a longer pitch than that seen in crystals of Rad51's prokaryotic homolog RecA, and places the ATPase site directly at a new interface between protomers. Although the filament exhibits approximate six-fold symmetry, alternate protein-protein interfaces are slightly different, implying that the functional unit of Rad51 within the filament may be a dimer. Additionally, we show that mutation of His352, which lies at this new interface, markedly disrupts DNA binding.

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In this report, the effects of osmotic pressure on BamHI cognate binding and catalysis were investigated and compared with a previous study on EcoRI (Robinson, C. R. and Sligar, S. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2186 -2191). Our observation of the dependence of binding and catalytic parameters on osmotic pressure has allowed for the comparison of hydration changes associated with site-specific DNA recognition for both endonucleases. Over a large range of osmotic pressures (), the dependence of BamHI on osmotic stress during cognate binding and catalysis was very different from that of the related endonuclease EcoRI. The binding of EcoRI to cognate DNA was dominated by a dehydration of the endonuclease-DNA complex, whereas binding by BamHI to its cognate sequence was accompanied by a solvent release corresponding to some 125 fewer waters. Catalytic analysis at elevated osmotic pressures indicated that both endonucleases had undergone a net hydration of the complex with BamHI displaying a much greater dependence on osmotic stress than EcoRI. Although the enzymes shared core structural motifs, comparisons of high resolution x-ray structures revealed many different secondary structural features of the complexed endonucleases. The large difference in hydration changes by both BamHI and EcoRI could be attributed to these dissimilar secondary structural features, as well as the functional differences of the two endonucleases during sitespecific DNA recognition.The BamHI endonuclease binds the DNA recognition sequence 5Ј-GGATCC-3Ј with remarkable specificity. The enzyme catalyzes double strand hydrolysis in the presence of divalent cations (Mg 2ϩ , Mn 2ϩ ) after the first guanine base, leaving staggered 4-base pair (bp) 1 overhangs (1-3). Previous studies that investigated the effects of increased osmotic stress on endonuclease specificity for both BamHI and EcoRI reveal that slight perturbations in water activity could cause the enzymes to lose specificity for their cognate DNA recognition sequences (4 -6). Hence, in the presence of cosolvents, BamHI and EcoRI have a propensity to cleave at non-cognate or "star" sites which are characterized by base pair (bp) 1 changes within the hexameric cognate recognition sequence. In these studies which employed added solutes to modulate the various colligative properties of the reaction solution, increase in the observed star activity was only correlated with osmotic pressure () and was not correlated with other solution properties, such as dielectric constant, viscosity, and mole fraction of water (4, 6). A key experiment in determining whether these effects were linked to changes in hydration was through the reversion of the observed star activity with the application of hydrostatic pressure (5, 6). Hydrostatic pressure has been shown to preferentially hydrate macromolecules and biomolecular complexes (7,8). The stringent molecular recognition of the endonucleases was restored by the application of hydrostatic pressure under star conditions, presumably through the ...

Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.