DNA replication in Escherichia coli is catalyzed by DNA polymerase III holoenzyme. This enzyme, thought to coordinate leading and lagging strand synthesis as an asymmetric dimer, contains polymerizing activity in a core plus essential auxiliary factors. Two of the auxiliary factors, tau and gamma, are the products of one gene, dnaX. They are synthesized in equimolar amounts and, although their exact functions are unknown, it is possible that their asymmetry. Tau is the translation product of the full-length dnaX reading frame; the shorter gamma shares the same N-terminal sequence as tau but is terminated within the tau reading frame by a programmed ribosomal frameshift followed by a nonsense condon in the new frame. Knowledge of the sequence and mechanism of gamma formation makes possible investigations of tau and gamma functions. Specifically, the requirement for each tau and gamma in vivo (because the in vitro data are equivocal) will be tested by generation of mutant dnaX alleles which synthesize only tau or only gamma and gene replacement. Other mutant genes and proteins altered in ATPase and suspected helicase motifs will be analyzed in vivo and in vitro to correlate loss of complementing activity, ATPase, helicase, and ability to support polymerization in vitro. Second, dnaX gene expression will be preliminarily characterized. Third, the sequenced of dnaA mutations which suppress dnaX(Ts) mutants will be determined.
