CHIMERIC DNA POLYMERASE--CONSTRUCTION & CHARACTERIZAT1ON

THIS PROJECT WILL CONSTRUCT, EXPRESS, AND CHARACTERIZE A SET OF HYBRID RECOMBINANT DNA POLYMERASES. THE HYBID DNA POLYMERASES WILL POSSESS A HIGHLY PROCESSIVE 5'-3' DNA POLYMERASE ACTIVITY DERIVED FROM THE LARGE DOMAIN OF T3 BACTERIOPHAGE GENE 5 AND A MODERATE 3'-5' EXONUCLEASE ACTIVITY DERIVED FROM THE SMALL DOMAIN OF THE KLENOW FRAGMENT. THIS WILL BE ACCOMPLISHED USING A NOVEL CHIMERIC CONSTRUCTION/EXPRESSION VECTOR. THE VECTOR WILL ALLOW THE RAPID GENERATION OF FOUR CHIMERIC CONSTRUCTS, EACH HAVING A DIFFERENT TRANSITION SITEBETWEEN THE DNA SEQUENCES ENCODING THE 3'-5' EXONUCLEASE ACTIVITY OF THE KLENOW FRAGMENT AND THE DNA SEQUENCES ENCODING THE 5'-3' T3 DNA POLYMERASE ACTIVITY. THE CHIMERICPOLYMERASES WILL BE A USEFUL MOLECULAR BIOLOGY REAGENT IN CIRCUMSTANCES THAT REQUIRE A HIGH DEGREE OF PROCESSIVITY ANDFIDELITY DURING THE DNA POLYMERIZATION REACTION. POTENTIAL APPLICATIONS INVOLVE THE IMPROVEMENT OF SITE-SPECIFIC MUTAGENESIS REACTIONS, DNA SEQUENCING, AND AMPLIFICATION OF LARGE GENES. IN ADDITION TO ENGINEERING THESE HYBRID POLYMERASE CONSTRUCTS, THE METHODOLOGIES DEVELOPED IN PHASE I OF THIS PROJECT WILL PROVIDE A VALUABLE MODEL SYSTEM TO RESEARCHERS FOR FUTURE HYBRID PROTEIN CONSTRUCTIONS. IN PHASE II, THE EXPERIENCE GAINED FROM THE METHODOLOGIES, CONSTRUCTION, AND ANALYSIS OF THE RECOMBINANT POLYMERASES WILL BE APPLIED TO ENGINEER OTHER MULTIFUNCTIONAL ENZYMES WITH IMPORTANT INDUSTRIAL, PHARMACEUTICAL, AND MOLECULAR BIOLOGY APPLICATIONS.