Structural adaptation of vertebrate endonuclease G for 5-hydroxymethylcytosine recognition and function

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The information encoded in DNA bases and various chemical modifications of the bases define different types of living organisms. The primary goal of this project is to determine how a DNA modification specific to vertebrate animals is used by a protein also found specifically in vertebrates, including humans, to help repair damaged DNA. The knowledge gained from this study will improve understanding of how seemingly small changes in the amino acid sequence of a protein through evolution can affect its structure and result in dramatic differences in how vertebrate versus invertebrate animals read and utilize modifications in their DNAs. In addition, this project will bring together biochemistry and art faculty to develop a new course on “Visual Communication in Science.” This course will engage science and art students in a collaborative environment to learn how to present scientific concepts and data in ways that are visually impactful and scientifically accurate and informative for a variety of audiences.5-Hydroxymethylcytosine (5hmC) is an epigenetic modification that serves multiple functions in vertebrates but has no known function in invertebrates. The project aims to determine how mouse endonuclease G (mEndoG) cleaves DNA at 5hmC sites in the context of four-stranded Holliday junctions to promote site-specific recombination. The roles of specific amino acids will be studied by swapping sequences between mEndoG and a worm homolog that does not cleave 5hmC-modified DNA. The resulting enzymes will be structurally characterized by X-ray crystallography and their functions examined by DNA binding and cleavage assays to test whether the sequence swaps result in corresponding changes in structures and functions between the vertebrate and invertebrate homologs. Finally, junction recognition by mEndoG will be studied to determine whether this enzyme is a 5hmC-specific junction resolvase or whether junctions mimic another substrate, such as the cross-over structures of DNA entering and exiting a nucleosome particle in chromatin fibers.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.