Title

Study of the mechanistic features of DNA replication restart in neisseria gonorrhoeae

Date of Award

2012

Degree Name

M.S. in Chemistry

Department

Department of Chemistry

Advisor/Chair

Advisor: Matthew E. Lopper

Abstract

Neisseria gonorrhoeae is the causative organism of gonorrheal infection. This pathogen shows remarkable resistance to the oxidative damaging agents released by the neutrophils. The Bacterial genome is one of the important targets of these agents and studies indicate that DNA replication restart pathways help in bypassing the hazardous effects of these oxidative agents. The DNA replication restart pathway helps to reload and restart the stalled or derailed replication machinery onto the DNA which is blocked by obstructions such as single stranded nicks, double stranded breaks, or oxidized bases. In N. gonorrhoeae, the primosome proteins PriA and PriB carry out the DNA replication restart process by forming a nucleoprotein complex with the DNA. PriA, a helicase protein, binds and partially unwinds the DNA at the fork where the replication machinery will be reloaded. PriB serves to stimulate PriAs DNA binding and unwinding activity. PriA, PriB and the DNA form a ternary nucleo-protein complex in this process. In, E. coli the PriB has a high affinity interaction with ssDNA and a low affinity interaction with PriA, while N. gonorrhoeae PriB has a high affinity interaction with PriA and a low affinity interaction with ssDNA. The first part of my research contributed to understand the features of this affinity reversal. I performed pull down experiments, DNA unwinding assays and ATP hydrolysis experiments using single point alanine mutants of PriB. From my work, I have provided evidence that in N. gonorrhoeae, PriBs ssDNA binding activity is not required for PriB stimulation of PriA helicase, unlike what is seen in E. coli. Evidence that PriA provides resistance against oxidative damaging agents bolsters the importance of DNA replication restart pathway for the survival of this disease causing bacteria. Developing antibiotics that target this pathway could be a vital event in the field of drug discovery research. In this process, we have developed an enzyme-based assay to use in high-throughput screening to identify inhibitors of the DNA replication restart pathway in N. gonorrhoeae. Initial screening of several thousand compounds from small molecule chemical libraries has produced several lead compounds. I worked with one of the lead compounds, CGS-15493, and here I report the mechanistic features of its inhibition of the DNA replication restart pathway.

Keywords

Neisseria gonorrhoeae Immunological aspects Research, DNA replication Antagonists Research

Rights Statement

Copyright 2012, author

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