DYNAMICS OF BACTERIOPHAGE-T4 DNA-POLYMERASE FUNCTION - IDENTIFICATIONOF AMINO-ACID-RESIDUES THAT AFFECT SWITCHING BETWEEN POLYMERASE AND 3'-]5'-EXONUCLEASE ACTIVITIES

Many DNA polymerases are multifunctional with the ability to replicate DNA as well as to proofread misincorporated nucleotides. Since polyme rase and 3' --> 5' exonuclease activities appear to reside in spatiall y distinct active centers, there must be some mechanism for coordinati ng replication with proofreading and for transferring DNA between the two active centers. We have designed a genetic selection scheme to iso late bacteriophage T4 mutant DNA polymerases that are defective in ''s witching'' between polymerase and exonuclease activities. Amino acid r esidues that affected active-site-switching were identified in four re gions of the T4 DNA polymerase: two regions in the proposed polymerase domain and two regions in the proposed exonuclease domain. Representa tive mutant DNA polymerases from each region were purified for biochem ical studies. We propose that amino acid substitutions identified by m utational analysis affect critical contacts between T4 DNA polymerase and DNA that are required for transfer of DNA between the polymerase a nd exonuclease active centers. (C) 1995 Academic Press Limited