REGULATION OF DNA REPLICATION ELONGATION IN BACTERIA

The major goal of this study is to investigate the molecular mechanism and physiological consequence of the regulation of DNA replication elongation in bacteria. Regulation of DNA replication is essential for efficient bacterial duplication and affects bacterial evolution by preventing mutagenesis. We have previously identified a replication control mechanism in bacteria: the stress-inducible nucleotide (p)ppGpp directly inhibitions the replication protein DNA primase, pausing the progression of the replication fork reversibly. This regulation is conserved in Gram-positive and Gram-negative bacteria. However, the mechanism by which bacteria achieve this regulation remains incompletely understood. Recently, we obtained results that suggest that (p)ppGpp binds to two distinct domains of primase to elicit two distinct effects that affect the initiation of priming, movement of the replicative helicase, and replication fidelity. Thus, this regulation has implications on both genome integrity as well as evolution.Through this research we will gain insight that may be employed to prevent bacterial evolution of antibiotic resistance.1. Investigate how (p)ppGpp regulates replication speed via its interaction with the HID (helicase interaction domain) of DNA primaseWe discovered that (p)ppGpp binds to two distinct domains on the primase DnaG: the active site on the RNA polymerase domain and the C-terminal helicase interaction domain (HID). The HID is important for coordination between primase and the replicative helicase, whose progression determines replication fork speed. We will fully characterize how (p)ppGpp binds the primase HID and evaluate its contribution to regulating replication speed.We will test the hypothesis that (p)ppGpp-primase HID interaction halts replication in the middle of the elongation phase during sudden stress and starvation, allowing non-disruptive restart upon release of the stress, thus preventing replication demise due to uncontrolled replication.This will reveal new insights into how stress, including antibiotic stress and nutrient starvation, affects bacterial cell cycle progression and genome integrity.2. Characterize how (p)ppGpp affects mutagenesis and evolution via its interaction with the RPD (RNA polymerase domain) of DNA primaseWe have found that (p)ppGpp not only has a strong inhibitory effect on primase activity, but also has a fundamental impact on primase fidelity: it prevents primase read through via mispairing, suggesting that (p)ppGpp promotes the fidelity of primase in generating primers. We will characterize the molecular mechanism of priming fidelity via (p)ppGpp via biochemistry. We will also determine the effects of (p)ppGpp regulation of primase on mutagenesis and replication fidelity in live bacteria, combining primase mutants and employing a mutation assay that allows us to identify multiple mutation signatures that can lead to antibiotic resistance in bacteria.This will characterize a pathway via which stress affects mutagenesis and bacterial evolution.