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)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM046330-23
Application #
8500332
Study Section
Special Emphasis Panel (NSS)
Program Officer
Reddy, Michael K
Project Start
1991-07-01
Project End
2017-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
23
Fiscal Year
2013
Total Cost
$595,102
Indirect Cost
$269,732
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Schwer, Beate; Khalid, Fahad; Shuman, Stewart (2016) Mechanistic insights into the manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-p-nitrophenylphosphate and branched RNA substrates. RNA 22:1819-1827
Maughan, William P; Shuman, Stewart (2016) Distinct Contributions of Enzymic Functional Groups to the 2',3'-Cyclic Phosphodiesterase, 3'-Phosphate Guanylylation, and 3'-ppG/5'-OH Ligation Steps of the Escherichia coli RtcB Nucleic Acid Splicing Pathway. J Bacteriol 198:1294-304
Chauleau, Mathieu; Jacewicz, Agata; Shuman, Stewart (2015) DNA3'pp5'G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition. Nucleic Acids Res 43:6075-83
Chauleau, Mathieu; Das, Ushati; Shuman, Stewart (2015) Effects of DNA3'pp5'G capping on 3' end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance. Nucleic Acids Res 43:3197-207
Maughan, William P; Shuman, Stewart (2015) Characterization of 3'-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5'-Phosphate Capping Activity of RtcB3. J Bacteriol 197:3616-24
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
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
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; Chakravarty, Anupam K; Remus, Barbara S et al. (2013) Rewriting the rules for end joining via enzymatic splicing of DNA 3'-PO4 and 5'-OH ends. Proc Natl Acad Sci U S A 110:20437-42
Chakravarty, Anupam K; Shuman, Stewart (2012) The sequential 2',3'-cyclic phosphodiesterase and 3'-phosphate/5'-OH ligation steps of the RtcB RNA splicing pathway are GTP-dependent. Nucleic Acids Res 40:8558-67

Showing the most recent 10 out of 35 publications