Interaction of single-stranded DNA with the second DNA-binding site of the RecA nucleoprotein filament

RecA first forms a filament on single-stranded DNA (ssDNA), thereby forming the first site for ssDNA binding and, simultaneously, the second site for binding double-stranded DNA (dsDNA). Then, the nucleoprotein filament interacts with dsDNA, although it can bind ssDNA as well. The resulting complex searches for homology sites and performs strand exchange between homologous DNA molecules. The interaction of various ssDNAs with the second DNA-recognizing site of RecA was studied by gradually increasing the structural complexity of the DNA ligand. Recognizing ssDNA with the second site, the protein interacts with each nucleotide of the ligand, forming contacts with both internucleotide phosphate groups and nitrogen bases. Pyrimidine oligonucleotides d(pC) _n and d(pT) _n interacted with the second site of the RecA filament more efficiently than d(pA) _n did. This was due to a more efficient interaction of the RecA filament with the 5′-terminal nucleotide of pyrimidinic DNA and to the difference in specific conformational changes of the nucleoprotein filament in the presence of purinic and pyrimidinic DNAs. A comparison of thermodynamic characteristics of DNA recognition at the first and second DNA-binding sites of the filament showed that, at n > 10, d(pC) _n and d(pN) _n were bound at the second site less tightly than at the first site. At n > 20, the second site bound d(pA) _n more efficiently than the first site. The difference in d(pN) _n affinity for the first and second sites increased monotonically with increasing n. Possible mechanisms of a RecA-dependent search for homology and DNA strand exchange are discussed.