Mechanism and Specificity of DNA Repair Dealkylases

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Genomic DNA undergoes spontaneous chemical modification (alkylation) whereby cellular or environmental reactive chemicals alter its structure and information content; therefore, cells require DNA repair pathways to detect and counteract these deleterious changes. This research will characterize the mechanism and specificity of a newly discovered pathway called direct repair in which a DNA repair dealkylase restores the correct nucleotide via a reaction involving multiple steps. By integrating trainees at all levels into the project, the PI will prepare individuals for future careers in STEM-related fields. Students will receive interdisciplinary training in genetic, biochemical, and biophysical techniques while gaining specific expertise in protein and nucleic acid biochemistry. A continued focus will be on recruiting students from underrepresented groups and outreach to the community, from elementary school all the way up to medical school, to incite a passion for basic sciences and to foster a diverse scientific community.The project identifies ALKB and ALKBH2 as representative examples of a broad superfamily of DNA repair oxidative dealkylases and addresses important unanswered questions about how these enzymes search for lesions in DNA, distinguish repair substrates from non-substrates, and maintain specificity for a broad range of substrates that complement other known DNA repair pathways. The central hypothesis is that the mechanism of ALKB enzymes has parallels to independently evolved DNA glycosylases that use different chemistry and different structural folds to overcome similar biological and physical challenges. The research will apply a combined approach of enzymology, transient kinetics, structure/function analysis, kinetic modeling, and genetics to understand the mechanisms by which ALKB and ALKBH2 find and repair DNA damage.This project is funded jointly by the Genetic Mechanisms Cluster in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Chemistry of Life Processes Program in the Division of Chemistry in the Directorate for Mathematical and Physical Sciences.