The DNA topoisomerase IB (ToplB) family includes the eukaryotic nuclear and mitochondrial ToplB enzymes, the poxvirus topoisomerases, mimivirus ToplB, and the poxvirus-like topoisomerases of bacteria. ToplB enzymes relax DNA supercoils by iteratively breaking and rejoining one strand of the DNA duplex through a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. Vaccinia ToplB, which displays stringent specificity for cleavage at the sequence 5'(C/T)CCTT], in the scissile strand, provides a highly instructive model system for mechanistic studies of the ToplB family. Its cleavage and religation transesterification reactions are driven by a constellation of four amino acid side chains (Arg130, Lys167, Arg223, His265) that catalyzes the attack of Tyr274 on the scissile phosphate to form the covalent intermediate and expel a 5'-0H "leaving strand". The active site is not preassembled in the apoenzyme;rather its assembly is triggered by recognition of the CCCTT target site. Our functional studies of ToplB mutants and DNA target modifications are contributing to a coherent model of the ToplB mechanism, and the likely steps in active site recruitment.
We aim to dissect in a comprehensive fashion the contributions of individual atomic contacts between the vaccinia ToplB and the DNA during active site assembly, transesterification, and supercoil relaxation. Our studies of DNA transesterification have inspired us to investigate site-specific and end-specific transesterification reactions of RNA and the enzymes that catalyze them. This is an emerging field of study that embraces many important biological phenomena, including host antiviral defense, RNA-based innate immunity, cellular stress responses, and tRNA splicing.

Public Health Relevance

Understanding the catalytic mechanism of ToplB is a high priority because: (i) ToplB is implicated in virtually every DNA transaction in higher eukarya;(ii) nuclear ToplB is the target of anticancer drugs that exert their cytotoxicity by perverting the cleavage-religation equilibrium;(iii) ToplB enzymes are encoded by many bacterial and viral pathogens, where they present untapped targets for anti-infective drug discovery.

Agency
National Institute of Health (NIH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM046330-24
Application #
8680247
Study Section
No Study Section (in-house review) (NSS)
Program Officer
Reddy, Michael K
Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2014
Total Cost
Indirect Cost
City
New York
State
NY
Country
United States
Zip Code
10065
Das, Ushati; Chauleau, Mathieu; Ordonez, Heather et al. (2014) Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends. Proc Natl Acad Sci U S A 111:11317-22
Yakovleva, Lyudmila; Shuman, Stewart (2013) Chemical mutagenesis of vaccinia DNA topoisomerase lysine 167 provides insights to the catalysis of DNA transesterification. Biochemistry 52:984-91
Das, Ushati; Shuman, Stewart (2013) 2'-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa. RNA 19:1355-62
Tanaka, Naoko; Chakravarty, Anupam K; Maughan, Bill et al. (2011) Novel mechanism of RNA repair by RtcB via sequential 2',3'-cyclic phosphodiesterase and 3'-Phosphate/5'-hydroxyl ligation reactions. J Biol Chem 286:43134-43
Chakravarty, Anupam K; Shuman, Stewart (2011) RNA 3'-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5'-monophosphate ends. J Biol Chem 286:4117-22
Jain, Ruchi; Poulos, Michael G; Gros, Julien et al. (2011) Substrate specificity and mutational analysis of Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease. RNA 17:1336-43
Tanaka, Naoko; Meineke, Birthe; Shuman, Stewart (2011) RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo. J Biol Chem 286:30253-7
Chakravarty, Anupam K; Smith, Paul; Shuman, Stewart (2011) Structures of RNA 3'-phosphate cyclase bound to ATP reveal the mechanism of nucleotidyl transfer and metal-assisted catalysis. Proc Natl Acad Sci U S A 108:21034-9
Tanaka, Naoko; Shuman, Stewart (2011) RtcB is the RNA ligase component of an Escherichia coli RNA repair operon. J Biol Chem 286:7727-31
Tanaka, Naoko; Smith, Paul; Shuman, Stewart (2011) Crystal structure of Rcl1, an essential component of the eukaryal pre-rRNA processosome implicated in 18s rRNA biogenesis. RNA 17:595-602

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