A conserved insertion in protein-primed DNA polymerases is involved in primer terminus stabilisation.
Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or alpha-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of phi29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3'-5' exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in phi29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.
Dufour
Emmanuelle
E
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Instituto de Biología Molecular Eladio Viñuela (CSIC), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Rodríguez
Irene
I
Lázaro
José M
JM
de Vega
Miguel
M
Salas
Margarita
M
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D013698:Templates, Genetic; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004259:DNA-Directed DNA Polymerase