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Differential proximity probing of two DNA binding sites in the Escherichia coli recA protein using photo-cross-linking methods.

The DNA strand-exchange reaction catalyzed by the Escherichia coli RecA protein occurs between the two DNA binding sites that are functionally distinct. Site I is the site to which a DNA molecule (normally single-stranded DNA) binds first; this first binding makes site II available for additional DNA-binding (normally double- stranded DNA). Photo-cross linking was employed to identify the amino acid residues located close to the bound DNA molecule(s). A ssDNA oligo containing multiple 5-iodouracil residues (IdU) was cross-linked to RecA by irradiation with a XeC1 pulse laser (308 nm), and the cross-linked peptides were purified and sequenced. To differentiate the two DNA binding sites, we used two protocols for making RecA-ssDNA complexes: (1) IdU-containing oligo was mixed with a stoichiometric excess of RecA, a condition which favors the binding of the oligo to site I, and (2) RecA was first allowed to bind to a nonphotoreactive oligo and then chased with the IdU-containing oligo, a condition which favors the binding of the IdU-oligo to site II. We observed that when RecA was in excess (site I probing), cross-linking occurred to Met-164 which is located in the disordered loop 1 of the RecA crystal structure [Story, R.M., Weber, I.T., & Steitz, T.A. (1992) Nature 355, 318-325]. When site II was probed, the majority of cross-linking occurred to Met-202 or Phe-203, located in loop 2. These results support the idea that, as predicted by Story and co-workers (1992), the disordered loops are involved in DNA binding. The results also suggest that the two sites are not only functionally but also physically distinct.