Structural Characterization of Cancer-Associated Human Endonuclease III-like Protein 1 Variants
Ek, Marenn
Ek, Marenn
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Abstract
Marenn Ek, Marcos B. Ngo, and Sylvie Doubli� Title: Structural Characterization of Cancer-Associated Human Endonuclease III-like Protein 1 Variants Human Endonuclease III-like protein 1 (NTHL1) is a highly conserved bifunctional DNA glycosylase that is responsible for the removal of oxidized pyrimidine lesions from DNA via the base excision repair pathway. In humans, an absence of NTHL1 function has been shown to increase the risk of carcinogenesis, likely to due to a reduction in the ability of cells to reverse the daily onslaught of oxidative damage. Therefore, germline variants of NTHL1 identified in the population are of interest due to their potential role in carcinogenesis in patients. One such identified variant is the substitution of a tyrosine residue at Asp239 (D239Y) in the enzyme active site, which has been shown to result in complete loss of function for the removal of at least seven known substrates of NTHL1. Two additional variants, R100C and P125L, have been identified in individuals predisposed to cancer, but are otherwise unstudied. The goal of this project is to determine the effect of these mutations on NTHL1 function at the biochemical and structural level to further an understanding of the risks posed to individuals expressing these phenotypes. Point mutations D239Y, R100C and P125L were generated in a ?63 NTHL1 construct that removes an intrinsically disordered region of the protein and improves the odds of crystallization. This construct was then expressed in E. coli and purified using nickel affinity, a heparin column, and gel filtration chromatography. SDS-Page was used to confirm sample purity. Dynamic light scattering was used to confirm there was no protein aggregation before beginning crystallization trials. Preliminary crystals were obtained for D239Y and P125L, and optimization of crystallization conditions is in progress in order to obtain diffraction-quality crystals.
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Date
1/1/2026
Student Status
Senior (Graduating in 2026)
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Poster
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Biochemistry
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College of Agriculture and Life Sciences
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Life Science