Binding of SSB and RecA protein to DNA-containing stem loop structures: SSB ensures the polarity of RecA polymerization on single-stranded DNA

Single-stranded DNA-binding proteins play an important role in homologous p airing and strand exchange promoted by the class of RecA-like proteins. It is presumed that SSB facilitates binding of RecA on to ssDNA by melting sec ondary structure, but direct physical evidence for the disruption of second ary structure by either SSB or RecA is still lacking. Using a series of oli gonucleotides with increasing amounts of secondary structure, we show that stem loop structures impede contiguous binding of RecA and affect the rate of ATP hydrolysis. The electrophoretic mobility shift of a ternary complex of SSBDNA-RecA and a binary complex of SSB-DNA are similar; however, the me chanism remains obscure. Binding of RecA on to stem loop is rapid in the pr esence of SSB or ATP gammaS and renders the complex resistant to cleavage b y HaeIII, to higher amounts of competitor DNA or low temperature. The elong ation of RecA filament in a 5' to 3' direction is halted at the proximal en d of the stem. Consequently, RecA nucleates at the loop and cooperative bin ding propagates the RecA filament down the stem causing its disruption. The pattern of modification of thymine residues in the loop region indicates t hat RecA monomer is the minimum binding unit. Together, these results sugge st that SSB plays a novel role in ensuring the directionality of RecA polym erization across stem loop in ssDNA. These observations have fundamental im plications on the role of SSB in multiple aspects of cellular DNA metabolis m.