Our long-term goals are to understand the mechanism and biological functions of DNA topoisomerase IB (ToplB). The ToplB family includes eukaryotic nuclear and mitochondrial ToplB, poxvirus and mimivirus topoisomerases, and the poxvirus-like topoisomerases of bacteria. ToplB enzymes relax DNA supercoils by breaking and rejoining one strand of the DNA duplex. They act via a transesterification mechanism involving a covalent DNA-(3'-phospho-tyrosyl)-enzyme intermediate. This laboratory uses vaccinia virus as a model system to study ToplB. The vaccinia-encoded ToplB is packaged within the virus particle, where it plays a critical role in replicative fitness by aiding viral mRNA synthesis. A distinctive feature of the poxvirus ToplB is its specificity in forming a covalent intermediate at a target sequence 5'-(C/T)CCTT. All poxvirus topos recognize this site, as does the homologous mimivirus ToplB enzyme. We hypothesize that DNA target recognition triggers the recruitment of catalytic amino acid side chains to form the ToplB active site.
An aim of this project is to elucidate at single-atom resolution the structural basis for DNA transesterification and target site specificity and to define the conformational steps for active site assembly and supercoil relaxation. This will be accomplished by an innovative multidisciplinary approach involving DNA chemistry, protein modification with non-natural amino acids, and single-molecule studies, along with """"""""classical"""""""" structure-guided mutagenesis and biochemistry. We also aim to dissect genetically which properties of vaccinia ToplB are important in vivo, by gauging the effects of biochemically characterized ToplB mutations on vaccinia virus replication. Relevance: Understanding the catalytic mechanism of ToplB is a high priority because: ToplB is implicated in virtually every DNA transaction in human cells;nuclear ToplB is the target of anticancer drugs that exert their cytotoxicity by perverting the cleavage-religation equilibrium;and ToplB enzymes are distributed widely in bacterial and viral pathogens, where they present untapped targets for mechanism-based anti-infective drug discovery. Exploitation of new molecular targets for treatment of poxvirus infections is a pressing issue, given the concerns that smallpox could be used as a bioterror weapon and the risk of complications of vaccinia infections if a prophylactic vaccination program is resumed.

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 #
5R37GM046330-19
Application #
7640806
Study Section
Virology - A Study Section (VIRA)
Program Officer
Santangelo, George M
Project Start
1991-07-01
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
19
Fiscal Year
2009
Total Cost
$578,913
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Munir, Annum; Shuman, Stewart (2017) Characterization of Runella slithyformis HD-Pnk, a Bifunctional DNA/RNA End-Healing Enzyme Composed of an N-Terminal 2',3'-Phosphoesterase HD Domain and a C-Terminal 5'-OH Polynucleotide Kinase Domain. J Bacteriol 199:
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
Cho, Jang-Eun; Huang, Shar-Yin N; Burgers, Peter M et al. (2016) Parallel analysis of ribonucleotide-dependent deletions produced by yeast Top1 in vitro and in vivo. Nucleic Acids Res 44:7714-21
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
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
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
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

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