DNA ligases are ubiquitous enzymes that catalyze the essential final step in DNA replication and repair - the conversion of DNA nicks into phosphodiester bonds. The joining of a 5' phosphate strand to a 3' hydroxyl strand at the nick entails three chemical steps: (i) ligase reacts with ATP or NAD to form a covalent intermediate (ligase-adenylate) in which AMP is linked to a lysine on the enzyme; (ii) the AMP is transferred from the ligase to the 5' phosphate end to form a DNA-adenylate intermediate; (iii) ligase catalyzes attack by the 3'OH of the nick on DNA-adenylate to join the two polynucleotides and liberate AMP. Our goal is to understand how ligase reaction chemistry is catalyzed and how ligase recognizes """"""""damaged"""""""" (i.e., nicked) DNA - using a eukaryotic virus DNA ligase as a model. Chlorella virus PBCV1 ligase is the smallest eukaryotic ATP-dependent ligase known (298-aa). It consists only of the catalytic core, unembellished by the large flanking domains that decorate cellular ligases. Nonetheless, Chlorella virus ligase sustains mitotic growth and DNA repair in yeast when it is the only ligase in the cell. As the minimal eukaryotic ligase, and one with an intrinsic nick-sensing function, the Chlorella virus enzyme presents an attractive target for structural and functional analysis. We have crystallized Chlorella virus DNA ligase and determined the structure of the covalent ligase-AMP reaction intermediate at 2.0 A resolution. Models of nick recognition and catalysis suggested by the ligase-AMP structure will be tested and clarified by the experiments outlined in this proposal.
Our specific aims are: (1) to identify by structure-based mutagenesis the important functional groups of DNA ligase; (2) to define the interface between ligase-adenylate and nicked DNA using footprinting and crosslinking methods; (3) to determine by crystallography the structure of ligase-adenylate bound at a DNA nick; and (4) to determine the """"""""ground-state"""""""" structure of ligase bound to ATP. The proposed experiments blend biochemistry, molecular genetics, and structural biology to elucidate how the chemical and conformational steps of the ligation pathway are coordinated. The findings will provide new insights into DNA damage recognition - an issue relevant to human health in light of the emerging genetic connections between DNA repair pathways and human cancer predisposition.
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