Type I DNA topoisomerases alter the topology of DNA by transiently breaking and rejoining single DNA strands. The eukaryotic family of type I enzymes, which includes the nuclear enzymes and the topoisomerases encoded by poxviruses, comprises a group of proteins with shared biochemical properties and catalytic mechanism. The involvement of these enzymes in DNA replication, genetic recombination, and transcription, and the fact that topoisomerase I is the target of the camptothecin class of antitumor drugs, mandates a more complete understanding of their mechanism of action. This laboratory is pursuing a combined biochemical and molecular genetic study of eukaryotic topoisomerase I function and pharmacology using vaccinia virus as a model system. Vaccinia is unique among eukaryotic DNA viruses in that it encapsidates a topoisomerase within the infectious virus particle. The viral topoisomerase is similar in its enzymatic properties to the cellular counterpart, but differs from the cellular enzyme in three respects: (i) the 314-amino acid vaccinia topoisomerase is considerably smaller than the cellular type I enzymes (765 to 972-aa); (ii) vaccinia topoisomerase cleaves DNA at a specific recognition element CCCTT; (iii) vaccinia topoisomerase is inherently resistant to camptothecin. In this proposal, we address the specificity of topoisomerase interaction with DNA, and describe novel ways to examine how this specificity might illuminate the role of the topoisomerase I in DNA recombination. A rational mutagenesis strategy is presented that will yield a comprehensive map of vaccinia topoisomerase function and primary protein structure. The hypothesis is made that resistance or sensitivity to camptothecin can be accounted for by defined subdomains of the cellular and viral topoisomerases that influence the interaction of the drug with the covalent enzyme-DNA intermediate. The model will be tested by engineering vaccinia topoisomerase chimeras, by substituting local """"""""patches"""""""" of viral sequence with residues unique to the cellular enzymes, in an effort to render the viral enzyme camptothecin sensitive. Mutational analysis of the topoisomerase will be pursued in parallel with efforts to solve the structure of the protein via X-ray crystallography.

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National Institute of General Medical Sciences (NIGMS)
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Experimental Virology Study Section (EVR)
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Sloan-Kettering Institute for Cancer Research
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Reed, Benjamin; Yakovleva, Lyudmila; Shuman, Stewart et al. (2017) Characterization of DNA Binding by the Isolated N-Terminal Domain of Vaccinia Virus DNA Topoisomerase IB. Biochemistry 56:3307-3317
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
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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