Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biochemistry

Supervisor

Junop, Murray S.

Abstract

Deinococci exhibit a remarkable resilience toward DNA damage through the actions of several unique proteins, including DdrA. Although DdrA is critical for damage resistance, little is known about its mechanism of action. Despite sharing sequence similarity with Rad52, DdrA has been reported to lack single-stranded DNA annealing activity. In order to better characterize DdrA, structural studies were undertaken with the primary objective of gaining insight into the mechanism by which DdrA functions. Significant progress was made toward elucidating the X-ray crystal structure; in particular, identifying suitable DdrA domain boundaries for successful expression, purification and crystallization. In addition, we demonstrate for the first time that DdrA mediates ssDNA annealing to levels comparable to Rad52 in vitro. Residues (K22 and K105) critical for ssDNA binding and annealing were identified and further used to demonstrate that DdrA mediates resistance to extreme levels of DNA damage through its ability to anneal ssDNA in vivo.


Summary for Lay Audience

D. radiodurans is a bacterium that was discovered in 1956 after surviving on a can of meat that had been exposed to intense doses of radiation. Typically, radiation kills organisms by destroying DNA, the molecule that all organisms require to maintain biological function. The fact that D. radiodurans was able to survive meant that the bacterium was either able to protect its DNA from damage in an extraordinary fashion or able to repair its DNA following damage in an extraordinarily efficient manner. Once it was established that both D. radiodurans and E. coli, a radiation-sensitive bacterium, accumulate DNA damage to the same extent, the latter was deemed to be true.

It was later determined that the unique resistance to radiation stems, in part, from a unique protein, known as DdrA. The protein was found to be “turned on” in the cell following DNA damage, providing correlative evidence that it is involved in repair. Furthermore, when the protein was eliminated from the cell, the organism became more radiation-sensitive, providing the first causative evidence that the protein is involved in repair. Prior to the publication of this thesis, it had been shown that DdrA is able to interact with DNA as one would expect for a protein required to repair DNA. However, beyond this, no further details regarding the exact role of DdrA were known. This thesis demonstrates that DdrA is capable of annealing DNA, which is an important aspect of genomic reconstruction following DNA damage. Furthermore, we have identified the exact regions of the protein, which are involved in this process. Most significantly, we have determined that without this contribution by DdrA, cells are sensitive to DNA damage, underscoring the importance of this phenomenon in the living organism. To figure out exactly how annealing takes place at an atomic level, we are now interested in figuring out what DdrA bound to protein looks like using a technique, known as X-ray crystallography. Significant progress to this end has been made in the lab and our current research efforts are aimed at bringing this task to completion.

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