Date of Award
2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
Sylvie Doublié
Abstract
Polymerase θ is a large A-family DNA polymerase (2,590 residues) that repairs double-strand breaks and has unique lesion bypass abilities. Pol θ possesses an unusual three domain architecture consisting of a helicase-like domain connected to the polymerase domain through a central linker domain. As pol θ is already larger than most polymerases, the presence of structural inserts in its polymerase domain make the protein even larger. These inserts, which are conserved in mammalian pol θ, may have evolutionary importance as lower eukaryotes lack some or all of the inserts observed in humans; interestingly, parasites like trypanosomatids have a polymerase that has unique inserts not found in humans.
The polymerase domain alone harbors three inserts. The role of inserts 1 and 2 has been investigated, and recent experiments by our lab suggest that removal of insert 3 increases polymerase extension efficiency. However, it remains unknown how pol θ uses insert 3 to regulate the efficiency of DNA synthesis. By structurally and biochemically interrogating the properties of a polymerase domain variant that lacks disordered insert 3, we aim to uncover the requirements for efficient DNA extension and how that activity is modulated by structural motifs in pol θ. Recently, the ability of pol θ to coordinate and extend single-stranded DNA substrates has been reported. X-ray diffraction data of a pol θ variant lacking insert 3 in complex with single-stranded DNA and incoming nucleotide were collected at the Diamond synchrotron at 4.4 Å. These preliminary crystallographic data hint at the possible location of the DNA hairpin in the active site of the human polymerase, which has not been modeled before. Additionally, binding affinities of the pol θ polymerase domain and two insert deletion variants were determined.
Pol θ is found in most eukaryotes, including trypanosomatid parasites. Sequence alignments indicate that the pol θ protein of the trypanosomatid T. cruzi (which causes chronic Chagas disease) contains the polymerase domain, albeit with no vestigial exonuclease domain, and lacks the N-terminal helicase-like domain. Biochemical characterization of T. cruzi pol θ along with in vivo cellular studies have led to a better understanding of what role pol θ is playing in these parasites. Ultimately, expression of T. cruzi pol θ in E. coli was unsuccessful. In vivo studies of the parasite hold promise but require more optimization before any conclusions can be drawn from the system. The observation of altered growth with overexpression T. cruzi pol θ in parasites indicates that this protein plays an important role in parasite biology, but a continued rigorous approach is required to know for certain.
Language
en
Number of Pages
145 p.
Recommended Citation
Drogalis, Lea Kimberly, "Functional Characterization Of The Polymerase Domain Of Polymerase Θ In Humans And T. Cruzi" (2025). Graduate College Dissertations and Theses. 1984.
https://scholarworks.uvm.edu/graddis/1984