Structural changes of the RecA filament upon binding of cofactors have been investigated by smallangle neutron scattering. Both ATP and ADP increased the helical pitch of the RecA homopolymer, which is observed to be 7 nm in the absence of any cofactor. The binding of ATP altered the pitch to 9 nm, whereas the binding of ADP only produced a pitch of 8.2 nm. The pitch determined for the RecA complex with the ATP analog adenosine 5'-[y-thioltriphosphate was similar to that found with ATP. Thus, at least three, somewhat different, RecA helical filamentous structures may form in solution. The binding of DNA to RecA did not alter the pitch significantly, indicating that the cofactor binding is the determining factor for the size of the helical pitch of the RecA filament. We also found that elongation of the helical pitch is a necessary, but not a sufficient condition, for the coprotease activity of RecA. The presence of acetate or glutamate ions is also required. The pitch of the ADP . RecA filament is in agreement with that found in the crystal structure. This correlation indicates that this structure corresponds to that of the ADP RecA filament in solution, although this is not the species active in recombination.Keywords: RecA ; LexA; small-angle neutron scattering; cofactor; RecA filament. the size of the helical pitch and the coprotease activity of the RecA filament, that the stretched filament is active, whereas the compact filament is inactive [23, 241. However, the size of the helical pitch of RecA with ADP as cofactor has not been examined.In this study, we investigate the size of the helical pitch of several RecA cofactor complexes in solution, by using smallangle neutron scattering (SANS). This technique, which has earlier been used to study can provide the average structure in true solution under various conditions. We show that both ADP and ATP enlarge the helical pitch of the RecA filament, but to different extents, ruling out the proposed simple two-state model. MATERIALS AND METHODSRecA and LexA repressors were prepared according to procedures described previously [27, 281. ATP, ADP, and ATP [S] were purchased from Boehringer Mannheim and used without further purification. The concentrations were determined spectrophotometrically using cZhl, = 14500 M-' cm -' for the nucleotides and czxo = 21 700 M-' cm-' for RecA [29].Samples for SANS measurements were prepared by dialysis of 100 pM RecA (in 20 mM sodium phosphate and 25 % glycerol) against a 50 times volume of 'H,O (Sigma 99% purity) buffer containing 20 mM sodium phosphate, 50 mM NaC1, 2 m M MgCl,, and 1 mM 2-mercaptoethanol, at 4°C. The Na,HPO,/NaH,PO, ratio was either 1 : 3 (pH 6.5) or 3 : 1 (pH 7.5) depending upon the desired pH. The dialysis buffer was changed three times during a total of 18 h. A small aliquot of cofactor (in 'H,O buffer) was subsequently added to RecA, to give a final

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA±DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA±DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5 H -O-3-thiotriphosphate or guanosine 5 H -O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA±DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.Keywords: RecA protein; homologous recombination; nucleotide cofactor; DNA binding; RecA filament.RecA protein is a cardinal element of the DNA repair system in Escherichia coli [1±3]. The protein regulates the synthesis of DNA repair enzymes (SOS induction), catalyzes homologous recombination and is involved in mutagenesis. Purified RecA mimics these activities in vitro: RecA catalyzes strand exchange between two homologous DNA molecules [4,5], stimulates autocleavage of LexA repressor [6,7] and UmuD protein [8], and interacts with UmuD H protein [9]. For these activities, RecA requires ATP or dATP as a cofactor and interacts with DNA with very high cooperativity to form a filamentous complex, in which RecA subunits are organized in a helical manner around the DNA [10,11]. The activation of RecA occurs only with ATP or dATP. Other natural nucleotides, ADP, CTP, GTP or TTP do not activate RecA [5,12±14], even though they interact with [13,15] and are hydrolyzed by RecA [14,16].Hydrolysis of the nucleotide is required neither for strand exchange [17] nor coprotease activity [12,13]. Both these reactions can be promoted by a nonhydrolysable analog, adenosine 5 H -O-3-thiotriphosphate (ATP [S]). The activation of RecA by ATP can be explained by an allosteric mechanism. The activating nucleotides, ATP and ATP [S], affect the conformation of RecA protein [18±20], and modify the structure of the RecA filament [11,21±23] and the DNA binding mode [11,24±26]. In the pr...

Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical filament independently of DNA. The sizes of the cross-sectional and helical pitch of the filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA filament, the structure of XRad51.1 filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleofilament should be very different from that on RecA.