The DNA polymerase III holoenzyme is the replicative complex of E. coli, responsible for the synthesis of the majority of the chromosome. This replicative complex exhibits many properties in common with other cellular replicases that distinguish it from simpler polymerases. These properties include a high rate of elongation, the ability to form an ATP-dependent highly processive clamp on the DNA template, and the ability to function as an asymmetric dimer with distinguishable leading and lagging strand polymerases. In this proposal, we focus on four issues related to the dual assembly roles that DnaX protein serves as the central organizational component for holoenzyme subunit assembly and as a catalytic ATPase that sets the beta sliding clamp on DNA, a requisite step in formation of replicative complexes: (1) The dnaX gene in E. coli encodes two products -- full-length tau and a shorter product, gamma, that results from programmed ribosomal frameshifting. The identification of gamma as a holoenzyme component has come into question since tau is proteolyzed by the OmpT protease to make a nearly identical protein. We will resolve this issue. (2) We will also study the equilibria between holoenzyme subunits and subassemblies. This information and kinetic subunit assembly data on the formation of the principle intermediates will permit a complete description of the assembly pathway for the DNA polymerase III holoenzyme. (3) The 5-protein DnaX-complex recognizes primer termini and transfers the beta sliding clamp to them in an ATP-dependent reaction. We will measure the equilibria of DnaX- complex with all of the components of the reaction in the absence of nucleotide and in the presence of ATP, ADP and analogs. Through understanding the linkage between ATP hydrolysis and preferential DnaX complex affinities, we will be able to propose a mechanism for DnaX-complex function. (4) We will exploit a biotinylated fusion protein approach recently developed in our lab to determine the limits of domains within the DnaX protein required for its multiple interactions and functions.
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