This MIRA proposal consolidates and extends diverse lines of inquiry into fundamental DNA and RNA transactions that were heretofore supported by four longstanding NIGMS grants. The goal of this research is: (i) to understand the mechanisms and structures of enzymes that perform nucleic acid synthesis, modification, and repair; and (ii) to elucidate factors that regulate these events. The project integrates diverse experimental approaches (microbiology, biochemistry, structural biology, genetics) and applies them to model systems ranging from viruses to bacteria to fungi. The principal themes are: (1) The chemical mechanism and structural basis for end recognition by polynucleotide ligases and mRNA capping enzymes that catalyze nucleotidyl transfer to 5' phosphorylated ends via a covalent enzyme-(lysyl- N?)?NMP intermediate. We will solve structures of exemplary ATP-dependent DNA ligases and capping enzymes as their step 1 Michaelis complexes with NTP and metal cofactors. We will clarify the specificity of the NHEJ ligase LigD for a 3'-monoribonucleotide nick and of capping enzyme for ppRNA. We will employ time- lapse crystallography to probe the role of metals in phosphodiester synthesis by ligases. (2) The structure, mechanism, and distinctive specificities of fungal tRNA splicing enzymes Trl1 (tRNA ligase) and Tpt1 (tRNA 2'-phosphotransferase) ? as paradigms of an RNA repair system essential for normal cell physiology and as promising targets for anti-fungal drug discovery. We will determine structures of Trl1 and Tpt1 from the human fungal pathogens Aspergillus fumigatus and Candida albicans in complexes with substrates, cofactors, and reaction intermediates. (3) The mechanism and distinctive target specificity of a eukaryal tRNA anticodon nuclease ?ribotoxin? (Pichia acaciae toxin; PaT) that underlies species self-nonself discrimination. We will determine the structure of PaT in complex with its substrate anticodon loop of tRNAGln(UUG). We will illuminate the basis for protective immunity by the Pichia acaciae antitoxin ImmPaT by solving the structure of a PaTImmPaT heterodimer. (4) The RNA polymerase II (Pol2) CTD code. The Pol2 CTD, consisting of tandem heptapeptides of consensus sequence Y1S2P3T4S5P6S7, is essential for viability because it recruits proteins that regulate transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation. By genetically manipulating the fission yeast CTD, and gauging effects on cell growth and gene expression, we: (i) educed structure-activity relations for each ?letter? of the code; and (ii) defined combinations of letters that comprise ?words? that are ?read? by cellular factors, and which govern specific expression programs. We focus here on the roles of CTD and transcription factor Pho7 in fission yeast phosphate homeostasis, a mechanism whereby phosphate-acquisition genes are repressed in phosphate-replete cells (in a manner dependent on CTD phospho-status), and activated in response to phosphate starvation (an event dependent on Pho7).

Public Health Relevance

The mechanistic and structural studies of enzymes involved in diverse nucleic acid transactions proposed for the MIRA award have relevance for human physiology and pathophysiology insofar as: (i) defects in DNA repair enzymes and repair pathways underlie human genetic diseases, including cancer and immune deficiencies; (ii) tRNA splicing enzymes Trl1 and Tpt1 are promising drug discovery targets for treatment of invasive fungal infections (e.g. Aspergillosis and Candidiasis); (iii) tRNA-cleaving ribotoxins have potential applications as antivirals and anticancer agents; and (iv) the Pol2 CTD code impacts virtually every phase of Pol2 transcription and cotranscriptional RNA maturation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZRG1)
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Barski, Oleg
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Sloan-Kettering Institute for Cancer Research
New York
United States
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Munir, Annum; Abdullahu, Leonora; Damha, Masad J et al. (2018) Two-step mechanism and step-arrest mutants of Runella slithyformis NAD+-dependent tRNA 2'-phosphotransferase Tpt1. RNA 24:1144-1157
Garg, Angad; Goldgur, Yehuda; Schwer, Beate et al. (2018) Distinctive structural basis for DNA recognition by the fission yeast Zn2Cys6 transcription factor Pho7 and its role in phosphate homeostasis. Nucleic Acids Res 46:11262-11273
Sanchez, Ana M; Shuman, Stewart; Schwer, Beate (2018) RNA polymerase II CTD interactome with 3' processing and termination factors in fission yeast and its impact on phosphate homeostasis. Proc Natl Acad Sci U S A 115:E10652-E10661
Munir, Annum; Banerjee, Ankan; Shuman, Stewart (2018) NAD+-dependent synthesis of a 5'-phospho-ADP-ribosylated RNA/DNA cap by RNA 2'-phosphotransferase Tpt1. Nucleic Acids Res 46:9617-9624