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Title: Mitochondrial DNA metabolism : organisation, structure, and replication stalling
Author: Bailey, L. J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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A mouse model was generated that accumulates multiple point mutations throughout the mitochondrial genome, due to an exonuclease deficient DNA polymerase. The work described here has focused on studying the structure of the mitochondrial genome of these mice and has shown that these mice suffer an increase in replication stalling. Breakage of stalled molecules at specific points, leads to the generation of a linear 11 kb DNA molecule and aborts replication. It is proposed here that these numerous rounds of futile replication lead to ‘cellular exhaustion’ and therefore the premature ageing phenotype of the mutator mouse. 5-15% of mtDNA molecules contain a third strand of DNA, 7S DNA, in the non-coding region, forming a displacement (D) loop structure. Here two-dimensional agarose gel electrophoresis and circular reverse transcription of PCR indicate that an additional strand of RNA is present in the NCR of the opposite orientation to 7S DNA. Therefore, this region may form a DNA-RNA bubble structure of D/R-loops rather than just a simple D-loop. Mitochondrial DNA is organised into nucleoid complexes with a number of DNA binding proteins. One of these proteins, ATAD3B, has been demonstrated to have a preference for binding to D-loop structures. Therefore, a fragment of recombinant ATAD3B has been studied to determine further details of its binding properties and inform future studies in living cells. This work has identified the Q149R polymorphism as a variant that increases the ability of the protein to bind single and double stranded DNA, with only minor affects on D-loop binding. It is proposed that this protein has a role in the regulation of mtDNA topology.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available