Metal activation of synthetic and degradative activities of phi 29 DNA polymerase, a model enzyme for protein-primed DNA replication.
Analysis of metal activation on the synthetic and degradative activities of phi 29 DNA polymerase was carried out in comparison with T4 DNA polymerase and Escherichia coli DNA polymerase I (Klenow fragment). In the three DNA polymerases studied, both the polymerization and the 3'----5' exonuclease activity had clear differences in their metal ion requirements. The results obtained support the existence of independent metal binding sites for the synthetic and degradative activities of phi 29 DNA polymerase, according with the distant location of catalytic domains (N-terminal for the 3'----5' exonuclease and C-terminal for DNA polymerization) proposed for both Klenow fragment and phi 29 DNA polymerase. Furthermore, DNA competition experiments using phi 29 DNA polymerase suggested that the main differences observed in the metal usage to activate polymerization may be the consequence of metal-induced changes in the enzyme-DNA interactions, whose strength distinguishes processive and nonprocessive DNA polymerases. Interestingly, the initiation of DNA polymerization using a protein as a primer, a special synthetic activity carried out by phi 29 DNA polymerase, exhibited a strong preference for Mn2+ as metal activator. The molecular basis for this preference is mainly the result of a large increase in the affinity for dATP.
Esteban
J A
JA
Centro de Biologia Molecular (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
Bernad
A
A
Salas
M
M
Blanco
L
L
D001665:Binding Sites; D003090:Coliphages / Q000201:enzymology / Q000235:genetics*; D004256:DNA Polymerase I / Q000737:chemistry; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004789:Enzyme Activation / Q000187:drug effects; D005092:Exonucleases / Q000737:chemistry; D006868:Hydrolysis; D008274:Magnesium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D008958:Models, Molecular; D013604:T-Phages / Q000201:enzymology / Q000235:genetics
D004279:DNA, Viral; D008345:Manganese; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D008274:Magnesium