Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization.
Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of phi 29 DNA polymerase in DNA polymerization ranged from 10(4) to 10(6). The efficiency of mismatch elongation was 10(5)-10(-6)-fold lower than that of a properly paired primer terminus. These factors indicate that DNA polymerization catalyzed by phi 29 DNA polymerase is a highly accurate process. Conversely, the insertion fidelity of protein-primed initiation was quite low, the insertion discrimination factor being about 10(2). Mismatch elongation discrimination was also rather low: mismatched terminal protein (TP).dNMP complexes were elongated from 2- to 6-fold more slowly than the correct TP.dNMP complex. Even more, the 3'-->5' exonuclease activity of phi 29 DNA polymerase was unable to act on the TP.dNMP initiation complex, precluding the possibility that a wrong dNMP covalently linked to TP could be excised and corrected. Therefore, protein-primed initiation can be predicted as a quite inaccurate reaction. The problem of maintaining the sequence at the DNA ends is discussed in the context of a recently described model for protein-primed initiation.
Esteban
J A
JA
Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma, Madrid, Spain.
Salas
M
M
Blanco
L
L
D017103:Bacillus Phages / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium