DNA Polymerase Alpha Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Screen [Enzymology]

May 18th, 2015 by Lindow, J. C., Dohrmann, P. R., McHenry, C. S.

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild type Pol III alpha and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III alpha, ablating interaction with the epsilon proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with tau binding. A third class mapped within the known beta binding domain, decreasing interaction with the beta(2) processivity factor. Surprisingly, mutations within the beta binding domain also ablated interaction with tau, suggesting a larger tau binding site than previously recognized.
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