Structural Basis for Specificity in the Poxvirus Topoisomerase

Summary Although smallpox has been eradicated from the human population, it is presently feared as a possible agent of bioterrorism. The smallpox virus codes for its own topoisomerase enzyme that differs from its cellular counterpart by requiring a specific DNA sequence for activation of catalysis. Here we present crystal structures of the smallpox virus topoisomerase enzyme bound both covalently and noncovalently to a specific DNA sequence. These structures reveal the basis for site-specific DNA recognition, and they explain how catalysis is likely activated by formation of a specific enzyme-DNA interface. Unexpectedly, thepoxvirusenzymeusesamajorgroovebinding a helix that is not present in the human enzyme to recognize part of the core recognition sequence and activate the enzyme for catalysis. The topoisomerase-DNA complex structures also provide a three-dimensional framework that may facilitate the rational design of therapeutic agents to treat poxvirus infections.

[1]  J. Champoux,et al.  The structure of the transition state of the heterodimeric topoisomerase I of Leishmania donovani as a vanadate complex with nicked DNA. , 2006, Journal of molecular biology.

[2]  A. Mondragón,et al.  Crystal Structure of a Bacterial Type IB DNA Topoisomerase Reveals a Preassembled Active Site in the Absence of DNA* , 2006, Journal of Biological Chemistry.

[3]  J. Stivers,et al.  Novel and Specific Inhibitors of a Poxvirus Type I Topoisomerase , 2006, Molecular Pharmacology.

[4]  W. Stec,et al.  Catalytic phosphoryl interactions of topoisomerase IB. , 2005, Biochemistry.

[5]  C. Dekker,et al.  Friction and torque govern the relaxation of DNA supercoils by eukaryotic topoisomerase IB , 2005, Nature.

[6]  Y. Pommier,et al.  Structures of three classes of anticancer agents bound to the human topoisomerase I-DNA covalent complex. , 2005, Journal of medicinal chemistry.

[7]  H. Fineberg,et al.  Discovery of antivirals against smallpox. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  Yves Pommier,et al.  Mechanisms of camptothecin resistance by human topoisomerase I mutations. , 2004, Journal of molecular biology.

[9]  S. Shuman,et al.  Remote phosphate contacts trigger assembly of the active site of DNA topoisomerase IB. , 2004, Structure.

[10]  B. Moss,et al.  Poxvirus DNA topoisomerase knockout mutant exhibits decreased infectivity associated with reduced early transcription , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[11]  Lance Stewart,et al.  The mechanism of topoisomerase I poisoning by a camptothecin analog , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Shuman,et al.  Proton Relay Mechanism of General Acid Catalysis by DNA Topoisomerase IB* , 2002, The Journal of Biological Chemistry.

[13]  J. Champoux,et al.  Novel insights into catalytic mechanism from a crystal structure of human topoisomerase I in complex with DNA. , 2000, Biochemistry.

[14]  S. Shuman,et al.  Catalytic mechanism of DNA topoisomerase IB. , 2000, Molecular cell.

[15]  J. Champoux,et al.  Structural flexibility in human topoisomerase I revealed in multiple non-isomorphous crystal structures. , 1999, Journal of molecular biology.

[16]  F. Bushman,et al.  DNA Contacts Stimulate Catalysis by a Poxvirus Topoisomerase* , 1999, The Journal of Biological Chemistry.

[17]  T. Tsuruo,et al.  Retroviral expression of a mutant (GLY‐533) human DNA topoisomerase I cDNA confers a dominant form of camptothecin resistance , 1999, International journal of cancer.

[18]  J. Champoux,et al.  Structural insights into the function of type IB topoisomerases. , 1999, Current opinion in structural biology.

[19]  S. Shuman.  Vaccinia virus DNA topoisomerase: a model eukaryotic type IB enzyme. , 1998, Biochimica et biophysica acta.

[20]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  Chonghui Cheng,et al.  A Catalytic Domain of Eukaryotic DNA Topoisomerase I* , 1998, The Journal of Biological Chemistry.

[22]  F. Bushman,et al.  Molluscum Contagiosum Virus Topoisomerase: Purification, Activities, and Response to Inhibitors , 1998, Journal of Virology.

[23]  Chonghui Cheng,et al.  Conservation of Structure and Mechanism between Eukaryotic Topoisomerase I and Site-Specific Recombinases , 1998, Cell.

[24]  W G Hol,et al.  A model for the mechanism of human topoisomerase I. , 1998, Science.

[25]  J. Champoux,et al.  Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA. , 1998, Science.

[26]  Deirdre C. Stam,et al.  Washington, D.C. , 1998 .

[27]  S. Shuman,et al.  Mechanism of DNA transesterification by vaccinia topoisomerase: catalytic contributions of essential residues Arg-130, Gly-132, Tyr-136 and Lys-167. , 1997, Nucleic acids research.

[28]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[29]  T. Harris,et al.  Vaccinia DNA topoisomerase I: evidence supporting a free rotation mechanism for DNA supercoil relaxation. , 1997, Biochemistry.

[30]  Chonghui Cheng,et al.  Mutational Analysis of 39 Residues of Vaccinia DNA Topoisomerase Identifies Lys-220, Arg-223, and Asn-228 as Important for Covalent Catalysis* , 1997, The Journal of Biological Chemistry.

[31]  S. Shuman,et al.  Histidine 265 Is Important for Covalent Catalysis by Vaccinia Topoisomerase and Is Conserved in All Eukaryotic Type I Enzymes* , 1997, The Journal of Biological Chemistry.

[32]  S. Shuman,et al.  Identification of contacts between topoisomerase I and its target DNA by site‐specific photocrosslinking. , 1996, The EMBO journal.

[33]  S. Shuman,et al.  Vaccinia topoisomerase binds circumferentially to DNA. , 1994, The Journal of biological chemistry.

[34]  S. Shuman,et al.  Mutational analysis of vaccinia DNA topoisomerase defines amino acid residues essential for covalent catalysis. , 1994, The Journal of biological chemistry.

[35]  E A Merritt,et al.  Raster3D Version 2.0. A program for photorealistic molecular graphics. , 1994, Acta crystallographica. Section D, Biological crystallography.

[36]  A. Mondragón,et al.  Crystal structure of the amino-terminal fragment of vaccinia virus DNA topoisomerase I at 1.6 A resolution. , 1994, Structure.

[37]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[38]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[39]  Eric Patterson,et al.  Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites , 1991, Nucleic Acids Res..

[40]  S. Shuman,et al.  Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. , 1990, The Journal of biological chemistry.

[41]  B. Moss,et al.  Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli. , 1988, The Journal of biological chemistry.

[42]  L. Matsumoto,et al.  Site-specific recombination intermediates trapped with suicide substrates , 1987, Cell.

[43]  R. Burgess,et al.  Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA. , 1984, Nucleic acids research.

[44]  D. J. Clarke,et al.  DNA Topoisomerases , 2009, Methods in Molecular Biology™.

[45]  G. D. Duyne.  A Structural View of Tyrosine Recombinase Site-Specific Recombination , 2002 .

[46]  J. Champoux.  DNA topoisomerases: structure, function, and mechanism. , 2001, Annual review of biochemistry.

[47]  G N Murshudov,et al.  Use of TLS parameters to model anisotropic displacements in macromolecular refinement. , 2001, Acta crystallographica. Section D, Biological crystallography.

[48]  R. Tirumalai,et al.  Similarities and differences among 105 members of the Int family of site-specific recombinases. , 1998, Nucleic acids research.

[49]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.