The Use of a Molecular Probe to Investigate the Details of PriA Helicase Function

Authors

Presenter(s)

Luke F. Bugada

Files

Description

During DNA replication in both eukaryotic and prokaryotic cells, the replication machinery (replisome) invariably encounters structural DNA damage, an event that can result in disbanding of the replisome and the creation of a collapsed replication fork. In order for DNA replication to continue, the replisome must be reloaded onto the DNA strand, a process that often begins with unwinding of double-stranded (duplex) DNA by the primosome protein PriA. Little is known about the mechanism through which PriA unwinds DNA and begins replisome recruitment. We seek to shed new light on this mechanism through the use of a PriA inhibitor, compound 0207. In our study, we attempt to determine the method of inhibition, the three-dimensional structure of the PriA•0207 complex, and the 0207 binding site through steady-state kinetics experiments, x-ray crystallography experiments, and mutagenesis assays. Data from the steady-state kinetics titrations show that compound 0207 acts through a mixed mode of inhibition and binds to the PriA•ATP, PriA•DNA, and PriA•ATP•DNA complexes with equal affinities. PriA crystals are being grown in the presence of compound 0207 in an attempt to solve the three-dimensional structure of the PriA•0207 complex using x-ray crystallography. Finally, mutagenesis assays are being used to search for the 0207 binding site on the surface of PriA. A docking simulation based on steric and electrostatic interactions was used to identify possible 0207 binding sites. Single alanine substitutions of PriA were generated, each with an alteration designed to inhibit the binding of compound 0207. The combined results of these experiments will provide a more complete understanding of the interactions between PriA and compound 0207, which will contribute to the overall goal of understanding the detailed mechanisms through which PriA catalyzes duplex DNA unwinding to initiate replication restart.

Publication Date

4-9-2016

Project Designation

Honors Thesis

Primary Advisor

Matthew E. Lopper

Primary Advisor's Department

Chemistry

Keywords

Stander Symposium project

Disciplines

Arts and Humanities | Business | Education | Engineering | Life Sciences | Medicine and Health Sciences | Physical Sciences and Mathematics | Social and Behavioral Sciences

Recommended Citation

"The Use of a Molecular Probe to Investigate the Details of PriA Helicase Function" (2016). Stander Symposium Projects. 731.
https://ecommons.udayton.edu/stander_posters/731