Genetic and biochemical studies of bacteriophage T4 DNA polymerase 3'-->5'-exonuclease activity.
DNA polymerase exonucleolytic proofreading is important in attaining high fidelity DNA replication. One of the most well characterized proofreading activities is the 3'-->5'-exonuclease activity of bacteriophage T4 DNA polymerase. We have used genetic analyses and protein sequence comparisons to Escherichia coli DNA polymerase I to identify amino acids in the N-terminal region of T4 DNA polymerase that are required for exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonuclease activity 10(2)-10(4)-fold in various in vitro assays and decreased DNA replication fidelity in vivo. DNA replication activity was also reduced for the exonuclease-deficient DNA polymerases in vitro and in vivo. Reduction in DNA replication appeared to be due primarily to the interdependence of T4 DNA polymerase replication and proofreading activities; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repair primer termini that are not suitable substrates for extension. Observations reported here provide further evidence in support of the proposal that DNA polymerases have distinct 3'-->5'-exonuclease and polymerase active sites.
Reha-Krantz
L J
LJ
Department of Genetics, University of Alberta, Edmonton, Canada.
Nonay
R L
RL
D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001704:Biopolymers; D017931:DNA Primers; D004261:DNA Replication / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011089:Polydeoxyribonucleotides / Q000378:metabolism
D001704:Biopolymers; D017931:DNA Primers; D011089:Polydeoxyribonucleotides; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases