Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd.
Degree: MS(M.S.), Biochemistry, 2010, U of Massachusetts : Masters
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
Advisors/Committee Members: Karsten Theis, Craig T. Martin.
Subjects/Keywords: Mfd; Transcription coupled repair; DNA repair; bacteria E. coli; Helicase; Biochemistry; Biology; Cell Biology; Molecular Biology; Structural Biology
to Zotero / EndNote / Reference
APA (6th Edition):
Hsieh, C. (2010). Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd. (Masters Thesis). U of Massachusetts : Masters. Retrieved from http://scholarworks.umass.edu/theses/506
Chicago Manual of Style (16th Edition):
Hsieh, Chih-heng. “Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd.” 2010. Masters Thesis, U of Massachusetts : Masters. Accessed December 05, 2019.
MLA Handbook (7th Edition):
Hsieh, Chih-heng. “Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd.” 2010. Web. 05 Dec 2019.
Hsieh C. Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd. [Internet] [Masters thesis]. U of Massachusetts : Masters; 2010. [cited 2019 Dec 05].
Available from: http://scholarworks.umass.edu/theses/506.
Council of Science Editors:
Hsieh C. Probing the Activation Mechanism of Transcription-Coupled Repair Factor Mfd. [Masters Thesis]. U of Massachusetts : Masters; 2010. Available from: http://scholarworks.umass.edu/theses/506