Structural Impact of the Leukemia Drug 1-β-d-Arabinofuranosylcytosine (Ara-C) on the Covalent Human Topoisomerase I-DNA Complex*

1-β-d-Arabinofuranosylcytosine (Ara-C) is a potent antineoplastic drug used in the treatment of acute leukemia. Previous biochemical studies indicated the incorporation of Ara-C into DNA reduced the catalytic activity of human topoisomerase I by decreasing the rate of single DNA strand religation by the enzyme by 2–3-fold. We present the 3.1 Å crystal structure of human topoisomerase I in covalent complex with an oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand. The structure reveals that a hydrogen bond formed between the 2′-hydroxyl of Ara-C and the O4′ of the adjacent −1 base 5′ to the damage site stabilizes a C3′-endo pucker in the Ara-C arabinose ring. The structural distortions at the site of damage are translated across the DNA double helix to the active site of human topoisomerase I. The free sulfhydryl at the 5′-end of the nicked DNA strand in this trapped covalent complex is shifted out of alignment with the 3′-phosphotyrosine linkage at the catalytic tyrosine 723 residue, producing a geometry not optimal for religation. The subtle structural changes caused by the presence of Ara-C in the DNA duplex may contribute to the cytotoxicity of this leukemia drug by prolonging the lifetime of the covalent human topoisomerase I-DNA complex.

[1]  A. Collins.  DNA damage in ultraviolet-irradiated HeLa and CHO-Kl cells examined by alkaline lysis and hydroxyapatite chromatography. , 1977, Biochimica et biophysica acta.

[2]  R. Hertzberg,et al.  Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. , 1985, The Journal of biological chemistry.

[3]  T. Yeates.  Simple statistics for intensity data from twinned specimens. , 1988, Acta crystallographica. Section A, Foundations of crystallography.

[4]  R. Hertzberg,et al.  On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex. , 1989, Biochemistry.

[5]  G. A. van der Marel,et al.  Molecular structure of a DNA decamber containing an anticancer nucleoside arabinosylcytosine: conformational perturbation by arabinosylcytosine in B-DNA. , 1991, Biochemistry.

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

[7]  D. Tenen,et al.  Acute myelogenous leukemia: current treatment and future directions. , 1992, The American journal of medicine.

[8]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[9]  Axel T. Brunger,et al.  Assessment of Phase Accuracy by Cross Validation: the Free R Value. Methods and Applications , 1993 .

[10]  J. Plavec,et al.  How do the aglycones drive the pseudorotational equilibrium of the pentofuranose moiety in C-nucleosides? , 1994 .

[11]  K. Gardner,et al.  Solution structure of a DNA dodecamer containing the anti-neoplastic agent arabinosylcytosine: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement. , 1994, Biochemistry.

[12]  H. Nash,et al.  Suicide substrates reveal properties of the homology-dependent steps during integrative recombination of bacteriophage λ , 1995, Current Biology.

[13]  H. Nash,et al.  A novel suicide substrate for DNA topoisomerases and site-specific recombinases. , 1995, Nucleic acids research.

[14]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[15]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[16]  S. Grant.  Modulation of ara-C induced apoptosis in leukemia by the PKC activator bryostatin 1. , 1997, Frontiers in bioscience : a journal and virtual library.

[17]  K. Kohn,et al.  Effects of Uracil Incorporation, DNA Mismatches, and Abasic Sites on Cleavage and Religation Activities of Mammalian Topoisomerase I* , 1997, The Journal of Biological Chemistry.

[18]  G. Ireton,et al.  Reconstitution of human topoisomerase I by fragment complementation. , 1997, Journal of molecular biology.

[19]  Y. Pommier,et al.  Induction of Topoisomerase I Cleavage Complexes by the Vinyl Chloride Adduct 1,N 6-Ethenoadenine* , 1998, The Journal of Biological Chemistry.

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

[21]  S. Grant,et al.  Ara-C: cellular and molecular pharmacology. , 1998, Advances in cancer research.

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

[23]  Y. Pommier,et al.  Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. , 1998, Biochimica et biophysica acta.

[24]  A. Burgin,et al.  Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates. , 1999, Virology.

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

[26]  F. Bushman,et al.  DNA contacts by protein domains of the molluscum contagiosum virus type-1B topoisomerase. , 1999, Virology.

[27]  Y. Pommier,et al.  Induction of Reversible Complexes between Eukaryotic DNA Topoisomerase I and DNA-containing Oxidative Base Damages , 1999, The Journal of Biological Chemistry.

[28]  R M Esnouf,et al.  Further additions to MolScript version 1.4, including reading and contouring of electron-density maps. , 1999, Acta crystallographica. Section D, Biological crystallography.

[29]  Y. Pommier,et al.  Induction of topoisomerase I cleavage complexes by 1-beta -D-arabinofuranosylcytosine (ara-C) in vitro and in ara-C-treated cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

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

[31]  Y. Pommier,et al.  Substitutions of Asn-726 in the Active Site of Yeast DNA Topoisomerase I Define Novel Mechanisms of Stabilizing the Covalent Enzyme-DNA Intermediate* , 2000, The Journal of Biological Chemistry.

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

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

[34]  J. Holden,et al.  DNA topoisomerases as anticancer drug targets: from the laboratory to the clinic. , 2001, Current medicinal chemistry. Anti-cancer agents.

[35]  Y. Pommier,et al.  Topoisomerase I-mediated DNA damage. , 2001, Advances in cancer research.

[36]  J Navaza,et al.  Implementation of molecular replacement in AMoRe. , 2001, Acta crystallographica. Section D, Biological crystallography.

[37]  A. Burgin,et al.  Synthesis and use of DNA containing a 5'-bridging phosphorothioate as a suicide substrate for type I DNA topoisomerases. , 2001, Methods in molecular biology.

[38]  Y. Pommier,et al.  8-Oxoguanine rearranges the active site of human topoisomerase I , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[39]  A. Burgin,et al.  Regulation of site-specific recombination by the C-terminus of λ integrase , 2002 .

[40]  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.