ORCID
https://orcid.org/0009-0003-9235-2328
Year
2025
Season
Fall
Paper Type
Master's Thesis
College
College of Arts and Sciences
Degree Name
Master of Science in Biology (MS)
Department
Biology
NACO controlled Corporate Body
University of North Florida. Department of Biology
Committee Chairperson
Dr. Grzegorz Ciesielski
Second Advisor
Dr. Szymon Ciesielski
Third Advisor
Dr. Judith Ochrietor
Department Chair
Dr. James Gelsleichter
Abstract
Mitochondrial DNA (mtDNA) replication is essential for cellular energy production and requires precise coordination among core replisome components, including the mitochondrial helicase Twinkle, DNA polymerase γ (Pol γ), and mitochondrial single-stranded DNA-binding protein (mtSSB). Although Twinkle is known to govern initiation and progression of mtDNA replication, the mechanisms that regulate its activity and coordinate transitions between abortive and processive replication remain poorly understood.
This study investigated both intrinsic and extrinsic mechanisms regulating Twinkle helicase activity. Biochemical helicase assays revealed that two intrinsic regulatory elements, the N-terminal zinc-binding domain (ZBD) and the C-terminal tail, function as autoinhibitory domains that restrain unwinding activity and are required for productive stimulation by mtSSB. Removal of either domain increased basal helicase activity while disrupting responsiveness to mtSSB, demonstrating that these elements fine-tune Twinkle function and mediate auxiliary factor communication.
In addition, the mitochondrial Hsp40 chaperone Tid1 was identified as a previously unrecognized auxiliary replisome factor. Biolayer interferometry and single-molecule imaging demonstrated that Tid1 binds preferentially to single-stranded and forked DNA structures resembling replication intermediates. Chromatin immunoprecipitation sequencing revealed specific Tid1 localization to key regulatory elements within the mitochondrial D-loop, including the origin of heavy-strand replication and termination-associated sequences. Functional assays showed that Tid1 enhances both Pol γ–mediated DNA synthesis and Twinkle helicase activity, supporting a direct role in replisome regulation.
Together, these findings establish a model in which mtDNA replication is regulated through coordinated intrinsic control of Twinkle and modulation by auxiliary factors such as mtSSB and Tid1. This dual regulatory framework provides new insight into how mitochondrial replication accuracy and genome maintenance are achieved.
Suggested Citation
Buchanan, Seth P., "Regulation of the activity of mtDNA helicase, Twinkle" (2025). UNF Graduate Theses and Dissertations. 1378.
https://digitalcommons.unf.edu/etd/1378