# Mechanisms of DNA and RNA transactions

> **NIH NIH R35** · SLOAN-KETTERING INST CAN RESEARCH · 2021 · $1,077,600

## Abstract

PROJECT SUMMARY: 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-
Nζ)–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 PaT·ImmPaT 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
C...

## Key facts

- **NIH application ID:** 10142494
- **Project number:** 5R35GM126945-04
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** Stewart H Shuman
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,077,600
- **Award type:** 5
- **Project period:** 2018-05-03 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10142494

## Citation

> US National Institutes of Health, RePORTER application 10142494, Mechanisms of DNA and RNA transactions (5R35GM126945-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10142494. Licensed CC0.

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