Involvement of the "linker" region between the exonuclease and polymerization domains of phi29 DNA polymerase in DNA and TP binding.
For several DNA-dependent DNA polymerases it has been shown that their synthetic and degradative activities are organized in two separated modules. The functional coordination required between them to accomplish successfully the replication process is provided by important contacts with the substrate contributed by residues coming from both modules. These domains are connected by a central "linker" region adjacent to the "YxGG/A" motif, the putative limit of the polymerization domain. We describe here the mutational analysis of phi29 DNA polymerase in several residues of this region, connecting the N- and C-terminal domains and conserved in DNA polymerases able to start replication by protein-priming. The mutant polymerases with the less conservative changes showed reduced DNA binding activity. Additionally, their TP binding capacity was reduced, affecting the TP-deoxynucleotidylation in the absence of template. Interestingly, the role of the residues studied here in DNA binding seems to be especially important to start replication, when the polymerase enters from the closed binary into the ternary complex. These results allow us to propose that this interdomain region of phi29 DNA polymerase is playing an important role for substrate binding including both DNA and TP.
Truniger
Verónica
V
Instituto de Biología Molecular "Eladio Viñuela" (CSIC), Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain.
Bonnin
Ana
A
Lázaro
José M
JM
de Vega
Miguel
M
Salas
Margarita
M
D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D005092:Exonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000378:metabolism*
D003838:Deoxyadenine Nucleotides; D011108:Polymers; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; C012873:2'-deoxy-5'-adenosine monophosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases