Degree Type

Thesis

Date of Award

2020

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Scott W Nelson

Abstract

DNA double strand breaks (DSBs) are the most harmful type of DNA lesions. The Mre11/Rad50 (MR) protein complex participates in DSB repair and DNA replication. Here, we have used the bacteriophage T4 MR complex (gp46/gp47) to investigate the nuclease and ATPase mechanisms of its components. Ensemble and smFRET exonuclease assays reveal how ATP enhances the processivity and translocation of the MR complex on the DNA substrate. The integrity of the dimer interface is essential for the structural and functional coupling of Mre11 and Rad50. The ATP-binding state of Rad50 appears to be communicated to Mre11 through a network of electrostatic interactions. These interactions are likely to be established by amino acid residues that extend from the nucleotide binding domain of Rad50 to the Rad50 binding domain of Mre11. The exonuclease assays and the kinetic model described here can be used in future studies of the MR complex and its interactions with substrates and partner proteins. The allosteric residues identified in this study also suggest an avenue for physiological and artificial regulation of the activities of the MR complex. A combination of the methods used for this study with other structural and biochemical techniques can provide a more comprehensive picture of how Mre11 and Rad50 cooperate to carry out their critical roles.

DOI

https://doi.org/10.31274/etd-20210114-146

Copyright Owner

Tibebe Antonios Teklemariam

Language

en

File Format

application/pdf

File Size

130 pages

Available for download on Wednesday, July 07, 2021

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