Involvement of residues of the 29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase.
Bacteriophage Φ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of Φ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized.
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), C/Nicolás Cabrera 1, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Alicia
A
del Prado
Laurentino
L
Villar
Miguel
M
de Vega
Margarita
M
Salas
D000596:Amino Acids / Q000737:chemistry; D017103:Bacillus Phages / Q000201:enzymology; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000096:biosynthesis; D019281:Dimerization; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D003838:Deoxyadenine Nucleotides; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; C012873:2'-deoxy-5'-adenosine monophosphate; D004259:DNA-Directed DNA Polymerase