Involvement of phage phi29 DNA polymerase and terminal protein subdomains in conferring specificity during initiation of protein-primed DNA replication.
To initiate phi29 DNA replication, the DNA polymerase has to form a complex with the homologous primer terminal protein (TP) that further recognizes the replication origins of the homologous TP-DNA placed at both ends of the linear genome. By means of chimerical proteins, constructed by swapping the priming domain of the related phi29 and GA-1 TPs, we show that DNA polymerase can form catalytically active heterodimers exclusively with that chimerical TP containing the N-terminal part of the homologous TP, suggesting that the interaction between the polymerase TPR-1 subdomain and the TP N-terminal part is the one mainly responsible for the specificity between both proteins. We also show that the TP N-terminal part assists the proper binding of the priming domain at the polymerase active site. Additionally, a chimerical 29 DNA polymerase containing the GA-1 TPR-1 subdomain could use GA-1 TP, but only in the presence of phi29 TP-DNA as template, indicating that parental TP recognition is mainly accomplished by the DNA polymerase. The sequential events occurring during initiation of bacteriophage protein-primed DNA replication are proposed.
Pérez-Arnaiz
Patricia
P
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Campus Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Longás
Elisa
E
Villar
Laurentino
L
Lázaro
José M
JM
Salas
Margarita
M
de Vega
Miguel
M
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics*; D001665:Binding Sites; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D018741:Replication Origin; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004259:DNA-Directed DNA Polymerase