Characterization of Hda-beta clamp functions beyond the regulatory inactivation of DnaA in Escherichia coli
Baxter, Jamie Clinton
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The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication ( oriC ) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. In this work, I present evidence to illustrate potential roles for Hda beyond RIDA. Utilizing chromatin immunoprecipitation (ChIP), I demonstrated that Hda can be found distributively throughout the chromosome, and that a recombinant form of the protein is proficient for specifically impairing replication activity of DNA damage polymerases II and IV in vitro. This work also identified conditions where hda is required for growth, allowing for a scorable phenotype by which to assay for suppression of loss of hda in vivo. Further, I used the mutant dnaN 159 strain, which expresses the β 59 clamp protein, to gain insight into how the actions of Hda are coordinated with replication. Elevated expression of Hda impeded growth of the dnaN 159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of –1 frame-shift mutations characteristic of Pol IV. Using the dnaN 159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Lastly, elevated expression of Hda and dnaN mutants bearing the G174A mutation demonstrate high degrees of resistance to chemotherapeutic ribonucleotide reductase (RNR) inhibitors hydroxyurea and guanazole. These findings demonstrate an ability of Hda to impair the activities of Pols II and IV, both in vivo and in vitro. The work also suggests that requirements of one of more Hda functions are essential for cell viability, among which RIDA may be dispensable. Finally, the ability of Hda and β clamp mutants to confer resistance to hydroxyurea and guanazole may present a novel mechanism for coordinating DNA replication with deoxyribonucleotide supply through regulation of RNR.