Off-campus UMass Amherst users: To download campus access theses, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this thesis through interlibrary loan.

Theses that have an embargo placed on them will not be available to anyone until the embargo expires.

Document Type

Campus Access

Degree Program

Biochemistry

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2010

Month Degree Awarded

September

Keywords

Mfd, Transcription coupled repair, DNA repair, bacteria E. coli, Helicase

Abstract

Cells dedicate tremendous amounts of energy to express essential genes for survival. During transcription, RNA polymerase (RNAP) actively scans the template strand of DNA, stalling when it meets DNA damages. Stalled RNAP prevents repair by the nucleotide excision repair pathway (NER); a sub-pathway of NER named transcription-coupled repair (TCR) resolve this problem by removing RNAP and recruiting repair proteins. In Escherichia coli, a TCR protein named “Mutation Frequency Decline” (Mfd) couples removal of RNAP through its motor activity with recruitment of the NER repair proteins. Mfd can be divided into two functional halves; the N-terminal region (MfdN, domains 1-3) is essential for NER protein recruitment, and the C-terminal region (MfdC, domains 4-7) is responsible for RNAP-interaction and motor activity. Data suggest Mfd undergoes large conformational movement to activate RNAP removal and repair protein recruitment. To study the activation mechanism of Mfd, we created several full-length “hyperactive” mutants by perturbing interactions between MfdN and MfdC. In all mutants, residue 79 in domain 1 is changed from aspartic acid to arginine (D79R), disrupting a key salt bridge interaction with arginine 804 in domain 6. The linker connecting MfdN and MfdC was made cleavable to allow separation of MfdN and MfdC, which enable us to study activities in equal molar concentration. We have studied the effect of the D79R mutation in vivo (cytotoxicity and UV sensitivity) and in vitro (enzyme activity and thermal stability), and demonstrate that this single residues change render the enzyme “hyperactive”. This confirms our model of activation: activation of Mfd results from breaking communication between MfdN and MfdC

First Advisor

Karsten Theis

Second Advisor

Craig T. Martin

Share

COinS