Presentation Title

Structural Investigation of Polymerase Theta Helicase-like Domain

Presenter's Name(s)

Scott H. Vanson, UVMFollow

Project Collaborators

Sylvie Doublié (Advisor), Brian Eckenroth, Kedar Moharana, April Averill

Abstract

DNA damaging events occur every day in every cell of all living organisms and result in several types of lesions, including oxidized bases, crosslinks, and double-stranded breaks (DSB). Human DNA polymerase θ (Pol θ) is a large, 290 kDa DNA repair enzyme and is the only known protein to contain both a polymerase domain and a helicase-like domain (HLD), connected by a disordered central domain, as one molecule. Pol θ is the key mediator of the error-prone DSB repair pathway, Theta-mediated End Joining. This enzyme has been identified as a potential therapeutic target as it may be conferring a survival advantage to subsets of homologous recombination (HR)-deficient cancers, which display elevated expression levels of Pol θ and correlate with poor prognoses. Interestingly, the HLD has repeatedly been shown to lack DNA unwinding activity despite its structural similarity to bona fide helicases. We have crystallized the HLD in the presence of DNA in an effort to visualize binding. We have also performed a fluorescence-based helicase assay with chimeras of the HLD and its closest relative, archaeal Hel308, to determine the basis for the HLD’s inability to unwind DNA.

Recombinant HLD (amino acids 1-894) was expressed and purified to homogeneity. Optimization of crystallization conditions yielded rectangular crystals, which form in the presence of a partially single-stranded 25 base DNA substrate and diffracted to approximately 3.4 Å. Preliminary models indicate subdomain rearrangement and a different oligomeric state when compared to the previously solved apo crystal structure. Efforts are underway to place the DNA molecule. Remarkably, the helicase assays indicate that a chimeric HLD is able to unwind DNA. An improved structural understanding of Pol θ HLD will guide efforts to design novel small-molecule inhibitors and potentially improve outcomes for patients with HR-deficient tumors.

Primary Faculty Mentor Name

Sylvie Doublié

Status

Graduate

Student College

Graduate College

Program/Major

Cellular, Molecular and Biomedical Sciences

Primary Research Category

Biological Sciences

Second College (optional)

Larner College of Medicine

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Structural Investigation of Polymerase Theta Helicase-like Domain

DNA damaging events occur every day in every cell of all living organisms and result in several types of lesions, including oxidized bases, crosslinks, and double-stranded breaks (DSB). Human DNA polymerase θ (Pol θ) is a large, 290 kDa DNA repair enzyme and is the only known protein to contain both a polymerase domain and a helicase-like domain (HLD), connected by a disordered central domain, as one molecule. Pol θ is the key mediator of the error-prone DSB repair pathway, Theta-mediated End Joining. This enzyme has been identified as a potential therapeutic target as it may be conferring a survival advantage to subsets of homologous recombination (HR)-deficient cancers, which display elevated expression levels of Pol θ and correlate with poor prognoses. Interestingly, the HLD has repeatedly been shown to lack DNA unwinding activity despite its structural similarity to bona fide helicases. We have crystallized the HLD in the presence of DNA in an effort to visualize binding. We have also performed a fluorescence-based helicase assay with chimeras of the HLD and its closest relative, archaeal Hel308, to determine the basis for the HLD’s inability to unwind DNA.

Recombinant HLD (amino acids 1-894) was expressed and purified to homogeneity. Optimization of crystallization conditions yielded rectangular crystals, which form in the presence of a partially single-stranded 25 base DNA substrate and diffracted to approximately 3.4 Å. Preliminary models indicate subdomain rearrangement and a different oligomeric state when compared to the previously solved apo crystal structure. Efforts are underway to place the DNA molecule. Remarkably, the helicase assays indicate that a chimeric HLD is able to unwind DNA. An improved structural understanding of Pol θ HLD will guide efforts to design novel small-molecule inhibitors and potentially improve outcomes for patients with HR-deficient tumors.