Structural and functional comparative study of the complexes formed by viral ø29, 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 single-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 bacteriophages, ø29, Nf and GA-1, form with single-stranded DNA. Under the experimental conditions used, ø29 and Nf single-stranded DNA-binding proteins are stable monomers in solution, while GA-1 single-stranded DNA-binding protein presents a hexameric state, as determined in glycerol gradients. The thermodynamic parameters derived from quenching measurements of the intrinsic protein fluorescence upon single-stranded DNA binding revealed (i) that GA-1 single-stranded DNA-binding protein occludes a larger binding site (n=51 nt/oligomer) than ø29 and Nf SSBs (n=3.4 and 4.7 nt/monomer, respectively); and (ii) that it shows a higher global affinity 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, these parameters justify the differences displayed by the GA-1 single-stranded DNA-binding protein and single-stranded DNA complex under the electron microscope, and the requirement of higher amounts of ø29 and Nf single-stranded DNA-binding proteins than of GA-1 SSB in gel mobility shift assays to produce a similar effect. The structural differences of the nucleoprotein complexes formed by the three single-stranded DNA-binding proteins with single-stranded DNA correlate with their different functional stimulatory effects in ø29 DNA amplification.
Gascón
I
I
Centro de Biologia Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autńoma, Cantoblanco, 28049-Madrid, Spain.
Gutiérrez
C
C
Salas
M
M
D001407:Bacillus; D017103:Bacillus Phages / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005784:Gene Amplification; D009698:Nucleoproteins / Q000648:ultrastructure; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D014157:Transcription Factors; D014764:Viral Proteins; C120819:p4G protein, Bacillus phage GA-1; C052951:3 protein, Bacillus phage Nf