Evidence for nucleotide-mediated changes in the domain structure of the recA protein of Escherichia coli.

We have used limited trypsin digestion as a means of investigating changes in the structural properties of recA protein accompanying the binding of different nucleoside triphosphates. The levels of four partial digestion products are greatly increased in digests of recA protein complexed with dTTP, dATP, ATP, or the ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). These bands (22, 19, and 17.5 kilodaltons) are absent or present at reduced levels in digests of recA protein alone. Unlike these nucleotides, all of which bind tightly to recA protein, nucleotides and analogues that bind poorly produce little or no change in the digestion pattern of recA protein. We have compared the rates of fragment accumulation in the presence of dTTP and show a saturable dependence on nucleotide concentration. Binding of single-stranded DNA to recA protein does not alter the pattern of digestion products compared to protein alone, and the digestion pattern of recA protein-DNA-ATP gamma S ternary complexes is similar to that of uncomplexed enzyme. We have used monoclonal antibody binding, high-performance liquid chromatography separation of peptides, and amino acid composition analyses to localize the regions of recA protein which are altered in their susceptibility to trypsin when nucleoside triphosphates are present. The results of these analyses indicate that the fragments arise from trypsin cutting at two or more sites near the middle of the primary sequence. These cleavage sites are more than 80-110 residues away from the site of photoaffinity labeling by 8-N3ATP (Tyr-264). Our results suggest that, in the presence of certain nucleotides, recA protein is organized into two stable structural domains.