Determinants of T4 DNA polymerase function

T4 DNA polymerase (T4 gp43) is an RNA-binding autogenous translational represser. We have cloned the DNA polymerase gene (gene 43) of phage RB69 (a distant relative of T4) and compared its biological, enzymological, and nucleic acid binding properties to those of the T4 enzyme. The two proteins have similar lengths (898 residues for T4 and 903 for RB69) and polymerase-3' exonuclease functions, but differ in their RNA binding specificities. T4 gp43 is specific to mRNA from its own genome, whereas RB69 gp43 can bind and repress mRNA from either T4 or RB69. We constructed a number of truncated and chimeric RB69-T4 gp43 species as well as point mutants in attempts to localize the RNA and DNA binding sites and catalytic domains of these single-chained replication enzymes. Also, RB69 gp43 has been crystallized by A. Sattar and W. Königsberg ( Yale Univ.) and structural information derived by i. Wang and T. Steitz (Yale Univ.). Structural models have guided the construction of gp43 variants. Some in-frame deletions and point mutations of both T4 and RB69 gp43 exhibit dominant lethal phenotypes that we used to show that the two gp43's have different replicon specificities. The replicon specificity of gp43 is contributed by at least two non-contiguous segments of this enzyme. Conservative and non-conservative amino acid substitutions at some putative polymerase (POL) active site residues inactivated the enzyme, others did not. The viable POL" mutants are being analyzed for defects in base selection fidelity. Similarly, exonuclease (EXO) motif mutants have been isolated and their defects in editing are being studied.