Importance of the N-terminal region of the phage GA-1 single-stranded DNA-binding protein for its self-interaction ability and functionality.
The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages phi 29 and Nf. In this work, the self-interaction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo cross-linking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of phi 29 and Nf. Three deletion mutant proteins have been characterized, Delta N19, Delta N26, and Delta N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein Delta N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins Delta N26 and Delta N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.
Gascón
Irene
I
Centro de Biologia Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Carrascosa
José L
JL
Villar
Laurentino
L
Lázaro
José M
JM
Salas
Margarita
M
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry* / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D007700:Kinetics; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004247:DNA