Regulation Of The DNA Damage Response By The DNA Polymerase Manager Protein UmuD

Abstract

Organisms experience DNA damage from environmental as well as endogenous sources. When bacterial cells experience DNA damage DNA and other stresses, the SOS response is induced, leading to upregulation of at least 50 genes in E. coli. Many of the genes whose expression is induced as part of the SOS response are responsible for DNA repair and cell cycle regulation. Another group of genes, specialized Y family DNA polymerases with the ability to replicate damaged DNA, play a role in tolerance to DNA damage at a potentially mutagenic cost. Multiple layers of regulation control the activity of these potentially mutagenic proteins. The function of Y family DNA polymerases is regulated by UmuD, a manager protein, and its cleaved form, UmuD'. The umuD gene products directly interact with both Y family polymerases as well as the beta processivity clamp. The goal of this study is to determine the conformation and dynamics of the umuD gene products in order to understand how they regulate the cellular response to DNA damage. We are using fluorescence resonance energy transfer (FRET) and hydrogen-deuterium exchange mass spectrometry (HXMS) to probe the conformations of UmuD and UmuD'. In HXMS experiments, backbone amide hydrogens that are solvent-accessible become labeled with deuterium over time, whereas those are are not accessible do not become labeled. Our HXMS results reveal that the N-terminal arm of UmuD, which is not present in the cleaved form UmuD', exhibits local partial unfolding. Residues that contact the N-terminal arm show large differences between UmuD and UmuD'. Additionally, there are substantial regions of stable conformation in both proteins. Complete characterization of UmuD and UmuD' dynamics is currently in progress.