Date of Award

1-1-2021

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (vi, 74 pages) : illustrations (some color)

Dissertation/Thesis Chair

Janice D Pata

Committee Members

Nilesh Banavali, Pan Li, Randall Morse, Jon Paczkowski

Keywords

Staphylococcus aureus, Staphylococcus aureus infections, Methicillin resistance, Drug resistance in microorganisms, DNA polymerases

Subject Categories

Biochemistry

Abstract

In this dissertation I provide a pre-steady-state kinetic characterization of an antibiotic-resistant mutant of a Staphylococcus aureus DNA polymerase. Staphylococcus aureus is one of the most common causes of infections in humans, and is widely known for its ability to acquire resistance to most antibiotics. Staphylococci infections pose a significant burden to the healthcare system and increase mortality, as more than 95% of Methicillin-Resistant S. aureus (MRSA) infections do not respond to first-line antibiotics. The limited treatment options for Staphylococci infections underscores the need for novel, alternative strategies. In this thesis, I discuss 6-anilinouracils (6-AU), a family of potent dGTP analogs that specifically inhibit DNA Polymerase III of gram-positive bacteria. PolC is the alpha subunit of DNA Polymerase III and the main C-family replicative polymerase in low G+C content Gram-positive bacteria such as S. aureus. Here we discuss a variant of PolC with a mutation of Phenylalanine to Leucine at position 1261 (PolCF1261L) that exhibits resistance to ME-EMAU, a 6-AU that inhibits wild type PolC (PolCWT). We created constructs of PolCWT and PolCF1261L and performed pre-steady-state kinetic characterization of both polymerases to elucidate the specificity of EMAU for PolC, as well as to determine the potential for resistance. We found that PolCWT and PolCF1261L demonstrate comparable affinities for DNA; however, PolCF1261L binds the incoming nucleotide (dGTP) five times more tightly than PolCWT and demonstrates 10-fold slower polymerization than WT. From our pre-steady-state kinetic analyses, we report a KD,appEMAU of 0.014 μM, konEMAU of 0.3425 μM-1s-1, and a koffEMAU of 0.006 s-1. This study demonstrates that EMAU is a potent, selective inhibitor of PolCWT and that this resistant variant of PolC, although it demonstrates increased affinity for the incoming nucleotide, has decreased fitness and thus mutant strains are unlikely to out-compete WT strains to propagate resistance.

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