Involvement of the TPR2 subdomain movement in the activities of phi29 DNA polymerase.
The polymerization domain of phi29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we have constructed a phi29 DNA polymerase mutant able to form a disulfide bond between the apexes of TPR2 and thumb to limit the mobility of TPR2. Biochemical analysis of the mutant led us to conclude that TPR2 moves away from the thumb to allow the DNA polymerase to replicate circular ssDNA. Despite the fact that no TPR2 motion is needed to allow the polymerase to use the terminal protein (TP) as primer during the initiation of phi29 TP-DNA replication, the disulfide bond prevents the DNA polymerase from entering the elongation phase, suggesting that TPR2 movements are necessary to allow the TP priming domain to move out from the polymerase during transition from initiation to elongation. Furthermore, the TPR2-thumb bond does not affect the equilibrium between the polymerization and exonuclease activities, leading us to propose a primer-terminus transference model between both active sites.
Rodríguez
Irene
I
Instituto de Biología Molecular Eladio Viñuela (CSIC), Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Cantoblanco, Madrid, Spain.
Lázaro
José María
JM
Salas
Margarita
M
de Vega
Miguel
M
D017103:Bacillus Phages / Q000201:enzymology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D009038:Motion; D009154:Mutation; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000378:metabolism
D004270:DNA, Circular; D004220:Disulfides; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases