Viral and Bacterial DNA Ligases

Sloan-Kettering Inst Can Research, New York NY

Investigators

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Abstract

DESCRIPTION (provided by applicant): DNA ligases are ubiquitous enzymes that catalyze an essential final step in DNA replication and repair - the conversion of DNA nicks into phosphodiester bonds. RNA ligases participate in breakage-repair pathways that underlie tRNA splicing, post-transcriptional RNA editing, and cellular stress responses. The DNA and RNA ligases seal 5'-PO4 and 3'-OH polynucleotide ends via three chemical steps: (i) ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-N6)-AMP intermediate; (ii) AMP is transferred from the ligase to the 5'-PO4 DNA or RNA strand to form a DNA/RNA-adenylate intermediate (AppDNA or AppRNA); (iii) ligase catalyzes attack by the 3'-OH on AppDNA/RNA to form a phosphodiester and release AMP. Our goals are to understand how ligase reaction chemistry is catalyzed, how ligases recognize damaged DNA or RNA ends, and how domain movements and active site remodeling are used to choreograph the end- joining pathway. We study these problems using three model systems: a eukaryal virus-encoded DNA ligase (Chlorella virus DNA ligase: ChVLig); a bacterial NAD+-dependent DNA ligase (E. coli LigA), and a viral ATP- dependent RNA ligase (T4 Rnl2). During the previous grant period, we determined the atomic structure of ChVLig-AMP bound at a 3'-OH/5'-PO4 nick and structures of LigA and T4 Rnl2 bound to their nicked polynucleotide-adenylate intermediates. These structures, and functional studies inspired by them, are revealing mechanistic principles shared by all DNA and RNA ligases, as well as the unique domain modules and substrate specificities that distinguish the various branches of the ligase superfamily. We have extended our interests in bacterial DNA ligases to two subfamilies of ATP-dependent strand joining enzymes (named LigD and LigC) that participate in a non-homologous end joining (NHEJ) pathway of bacterial DNA repair. LigC and LigD are unique among known ligases in that they require a 3'-OH monoribonucleotide in order to perform efficient nick sealing. LigD is doubly unique insofar as it is a multifunctional enzyme composed of three autonomous catalytic domains: a ligase (LIG); a polymerase (POL), and a phosphoesterase (PE). The POL and PE domains comprise a suite of DNA end-healing activities that remodel the 3' terminus of the DSB prior to sealing by the LIG component. We propose a multidisciplinary agenda (blending biochemistry, molecular genetics, and structural biology) to tackle a next generation of issues in the field. Our specific aims are: (i) to exploit the protein-DNA structures we've solved to guide a mutational analysis of amino acids at the ligase-DNA interface; (ii) to probe the mechanism of adenylate transfer to lysine, via structural methods and chemical mutagenesis - an approach that circumvents the limitations to the genetically programmable protein tool kit; (iii) to solve the structure of the LigD phosphoesterase domain, which exemplifies a new family of 3' end-modifying enzymes; and (iv) to illuminate the distinctive substrate preference of bacterial NHEJ ligases for a 3'-OH monoribonucleotide nick. We are confident that the experiments we propose will yield new insights to phosphoryl transfer reaction mechanisms, nucleic acid damage recognition, and the evolution of nucleic acid repair systems.