Evidence for Elongation of the Helical Pitch of the RecA Filament Upon ATP and ADP Binding Using Small‐Angle Neutron Scattering

Abstract

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

Locations of Functional Domains in the RecA Protein

“…Structural intormation about RecA and the RecA-DNA filaments has been obtained mainly from electron microscopy studies (DiCapua et al, 1982;Williams and Spengler, 1986;Heuser and Oriffith, 1989;Egelman, 1993) and small-angle neutron scattering (SANS) measurements (DiCapua et al, 1989(DiCapua et al, , 1992Ellouze et al, 1995). The monomer RecA subunits in the filament are organized in a helical manner with a periodicity of about 6 subunits/turn both in the presence and absence of DNA (DiCapua et al, 1982;Williams and Spengler, 1986).…”

Arrangement of RecA protein in its active filament determined by polarized-light spectroscopy

“…The three-dimensional structure of RecA itself and in complex with ADP has been determined by x-ray crystallography (11), but the structure of the active RecA-DNA-ATP complex is not yet known at the atomic level. EM as well as small-angle neutron scattering (SANS) observations indicate significant structural variations between pure RecA, as helically stacked in crystal, and in its helical filament when binding ATP and DNA (10,12,13).We now focus on the organization and conformation of the RecA subunits of the fiber by analyzing the LD signals from a set of aromatic residues (Tyr and Trp) of the protein. These residues absorb light in the near UV region and provide LD signals that reveal their orientations relative to the filament axis.…”

“…The three-dimensional structure of RecA itself and in complex with ADP has been determined by x-ray crystallography (11), but the structure of the active RecA-DNA-ATP complex is not yet known at the atomic level. EM as well as small-angle neutron scattering (SANS) observations indicate significant structural variations between pure RecA, as helically stacked in crystal, and in its helical filament when binding ATP and DNA (10,12,13).…”

Gly-103 in the N-terminal Domain of Saccharomyces cerevisiae Rad51 Protein Is Critical for DNA Binding

“…ATP␥S, ADP-AlF 4 Ϫ ) are essential cofactors in filament formation. ATP and ADP significantly regulate the conformation of the filament and affect its pitch (18,20). Similar to RecA, eukaryotic and archaeal Rad51/ Dmc1 also adopt ring or filament structures in solution depending on the presence of DNA and ATP (21)(22)(23)(24)(25).…”