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. .

Public Health Relevance

Ligases are attractive targets for antimicrobial drug discovery. Inhibitors of bacterial NAD+-dependent DNA ligase (LigA) are promising candidates for broad-spectrum antibacterial therapy, given that: (i) NAD+- dependent ligases are present in all bacteria and are essential for bacterial growth in all cases studied, and (ii) LigA enzymes are structurally conserved among bacteria, but display unique substrate specificity and domain architecture compared to the ATP-dependent ligases of humans and other mammals. Our structure of E. coli LigA in complex with AppDNA inspires a strategy for inhibitor design. The LigA structure reveals a through-and-through """"""""tunnel"""""""" - from the exterior surface of LigA to the adenosine- binding pocket - that completely exposes the edge of the adenine base. In particular the adenine C2 atom is pointed directly into the tunnel, which is formed by a cage of hydrophobic amino acids. This tunnel is present in all LigA enzymes. In contrast, there is no such tunnel emanating from the adenosine binding pockets of human DNA ligase or Chlorella virus ligase. This situation invites the structure-based design of C2-substituted derivatives of adenosine (or non-nucleotide mimics thereof) as unique and selective inhibitors of LigA. One such compound, 2-methyladenosine, has excellent antimicrobial activity against Mycobacterium tuberculosis, in culture and within human macrophages. There is now a pressing need for new antibiotics against human tuberculosis, as available treatment options degrade with the emergence of multi-drug- resistant strains. This is a serious public health problem. We expect our studies of LigA structure and mechanism will stimulate the discovery of new compounds that either interdict LigA binding to NAD+ or nicked DNA, or """"""""poison"""""""" the ligation pathway by trapping a """"""""toxic"""""""" nicked-adenylate intermediate. Similar considerations - a unique structural domain and distinctive nucleic acid substrate specificity - recommend Rnl2-type RNA ligases as targets for drug development for treatment of infectious diseases caused by protozoan parasites, specifically trypanosomiasis (African sleeping sickness and Chagas disease) and leishmaniasis. .

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics C Study Section (MGC)
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Hagan, Ann A
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Sloan-Kettering Institute for Cancer Research
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Unciuleac, Mihaela-Carmen; Goldgur, Yehuda; Shuman, Stewart (2017) Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases. Proc Natl Acad Sci U S A 114:2592-2597
Schmier, Brad J; Chen, Xinguo; Wolin, Sandra et al. (2017) Deletion of the rnl gene encoding a nick-sealing RNA ligase sensitizes Deinococcus radiodurans to ionizing radiation. Nucleic Acids Res 45:3812-3821
Chauleau, Mathieu; Shuman, Stewart (2016) Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA). Nucleic Acids Res 44:2298-309
Chauleau, Mathieu; Shuman, Stewart (2013) Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3'-OH base mispairs and damaged base lesions. RNA 19:1840-7
Das, Ushati; Shuman, Stewart (2013) Mechanism of RNA 2',3'-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase. Nucleic Acids Res 41:355-65
Natarajan, Aswin; Dutta, Kaushik; Temel, Deniz B et al. (2012) Solution structure and DNA-binding properties of the phosphoesterase domain of DNA ligase D. Nucleic Acids Res 40:2076-88
Das, Ushati; Smith, Paul; Shuman, Stewart (2012) Structural insights to the metal specificity of an archaeal member of the LigD 3'-phosphoesterase DNA repair enzyme family. Nucleic Acids Res 40:828-36
Samai, Poulami; Shuman, Stewart (2012) Kinetic analysis of DNA strand joining by Chlorella virus DNA ligase and the role of nucleotidyltransferase motif VI in ligase adenylylation. J Biol Chem 287:28609-18
Smith, Paul; Nair, Pravin A; Das, Ushati et al. (2011) Structures and activities of archaeal members of the LigD 3'-phosphoesterase DNA repair enzyme superfamily. Nucleic Acids Res 39:3310-20
Samai, Poulami; Shuman, Stewart (2011) Functional dissection of the DNA interface of the nucleotidyltransferase domain of chlorella virus DNA ligase. J Biol Chem 286:13314-26

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