Structural and functional comparative study of the complexes formed by viral circle divide 29, Nf and GA-1 SSB proteins with DNA

Single-stranded DNA-binding proteins have in common their crucial roles in DNA metabolism, although they exhibit significant differences in their sing le-stranded DNA binding properties. To evaluate the correlation between the structure of different nucleoprotein complexes and their function, we have carried out a comparative study of the complexes that the single-stranded DNA-binding proteins of three related baeteriophages, o29, Nf and GA-1, for m with single-stranded DNA. Under the experimental conditions used, o29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, w hile GA-1 single-stranded DNA-binding protein presents a hexameric state, a s determined in glycerol gradients. The thermodynamic parameters derived fr om quenching measurements of the intrinsic protein fluorescence upon single -stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding pr otein occludes a larger binding site (n = 51 nt/oligomer) than o29 and Nf S SBs (n = 3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a af finity for single-stranded DNA (GA-1 SSB, K-eff = 18.6 x 10(5) M-1; o29 SSB , K-eff = 2.2 x 10(5) M-1; Nf SSB, K-eff = 2.9 x 10(5) M-1). Altogether, th ese parameters justify the differences displayed by the GA-I single-strande d DNA-binding protein and single-stranded DNA complex under the electron mi croscope, and the requirement of higher amounts of o29 and Nf single-strand ed DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to pr oduce a similar effect. The structural differences of the nucleoprotein com plexes formed by the three single-stranded DNA-binding proteins with single -stranded DNA correlate with their different functional stimulatory effects in o29 DNA amplification. (C) 2000 Academic Press.