GENETIC AND BIOCHEMICAL-STUDIES OF BACTERIOPHAGE-T4 DNA-POLYMERASE 3'-]5'-EXONUCLEASE ACTIVITY

DNA polymerase exonucleolytic proofreading is important in attaining h igh fidelity DNA replication. One of the most well characterized proof reading activities is the 3'-->5'-exonuclease activity of bacteriophag e T4 DNA polymerase. We have used genetic analyses and protein sequenc e comparisons to Escherichia coli DNA polymerase I to identify amino a cids in the N-terminal region of T4 DNA polymerase that are required f or exonucleolytic proofreading. Mutant DNA polymerases with amino acid substitutions D112A/E114A, D219A, or D324A reduced 3'-->5'-exonucleas e activity 10(2)-10(4)-fold in various in vitro assays and decreased D NA replication fidelity in vivo. DNA replication activity was also red uced for the exonuclease-deficient DNA polymerases in vitro and in viv o. Reduction in DNA replication appeared to be due primarily to the in terdependence of T4 DNA polymerase replication and proofreading activi ties; T4 DNA polymerase requires 3'-->5'-exonuclease activity to repai r primer termini that are not suitable substrates for extension. Obser vations reported here provide further evidence in support of the propo sal that DNA polymerases have distinct 3'-->5'-exonuclease and polymer ase active sites.