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• W2052555812 abstract "1-ॆ-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.1NH3 1-ॆ-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.1NH3 ॆ-d-arabinofuranosylcytosine root mean square deviation protein data bank Human topoisomerase I solves the DNA topological problems that arise from a wide variety of nuclear processes including replication, transcription, and recombination (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar). The enzyme nicks one strand of duplex DNA using a transesterification reaction that produces a transient 3′-phosphotyrosine linkage and guides the relaxation of either positive or negative superhelical tension by a proposed 舠controlled rotation舡 mechanism (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar). The enzyme then catalyzes a second transesterification in which the free hydroxyl at the 5′-end of the nicked DNA strand attacks the phosphotyrosine bond, resealing the nick, and releasing a more relaxed DNA molecule. Topoisomerase I plays a vital role in maintaining DNA stability and is known to travel with active replication and transcription complexes in human cells (4Pommier Y. Pourquier P. Fan Y. Strumberg D. Biochim. Biophys. Acta. 1998; 1400: 83-105Google Scholar,5Holden J.A. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 1-25Google Scholar).Human topoisomerase I is the sole target of the camptothecins (CPT), a potent class of anticancer drugs used to treat late-term solid malignancies (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 4Pommier Y. Pourquier P. Fan Y. Strumberg D. Biochim. Biophys. Acta. 1998; 1400: 83-105Google Scholar, 6Hsiang Y.H. Hertzberg R. Hecht S. Liu L.F. J. Biol. Chem. 1985; 260: 14873-14878Google Scholar). Camptothecin effectively targets the religation phase of topoisomerase I catalysis by stabilizing the covalent protein-DNA complex and trapping the enzyme on DNA (7Hertzberg R.P. Caranfa M.J. Hecht S.M. Biochemistry. 1989; 28: 4629-4638Google Scholar, 8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar). In this way, CPT converts topoisomerase I into a cellular poison. Human topoisomerase I is also affected by several forms of DNA damage, including abasic lesions, wobble base pairs, and base pair mismatches (9Pourquier P. Ueng L.M. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Google Scholar, 10Pourquier P. Bjornsti M.A. Pommier Y. J. Biol. Chem. 1998; 273: 27245-27249Google Scholar, 11Pourquier P. Ueng L.M. Fertala J. Wang D. Park H.J. Essigmann J.M. Bjornsti M.A. Pommier Y. J. Biol. Chem. 1999; 274: 8516-8523Google Scholar, 12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar). Such lesions can impact each stage of topoisomerase I's catalytic cycle, including DNA binding, single-strand DNA cleavage, and religation.1-ॆ-d-Arabinofuranosylcytosine (Ara-C)1 is a nucleoside analogue used in the treatment of acute leukemia (14Grant S. Adv. Cancer Res. 2002; 72: 197-233Google Scholar, 15Mastrianni D.M. Tung N.M. Tenen D.G. Am. J. Med. 1992; 92: 286-295Google Scholar). Ara-C and the standard cytosine DNA base differ by the presence of a 2′-hydroxyl on the arabinose ring of the drug (Fig.1). Ara-C is thought to inhibit DNA polymerases central to replication and repair processes, and thus to slow the growth of malignant cells (16Collins A.R.S. Biochim. Biophys. Acta. 1977; 478: 461-473Google Scholar, 17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar). The detailed impact of Ara-C on human cells, however, is poorly understood. Incorporation of Ara-C into DNA causes localized alterations in the DNA duplex, including changes in sugar pucker, base stacking, and backbone torsion angles (17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 18Gao Y.-G. van der Marel G.A. van Boom J.H. Wang A.H.J. Biochemistry. 1991; 30: 9922-9931Google Scholar). Biochemical studies using human topoisomerase I have revealed that Ara-C incorporation at the +1 position of the intact (non-scissile) strand adjacent to the site of single-strand DNA cleavage induces a 4–6-fold increase in covalent topoisomerase I-DNA complexes caused by a 2–3-fold decrease in the rate of religation by the enzyme (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). Because the stabilization of covalent topoisomerase I-DNA complexes converts the enzyme into a cellular poison, the cytotoxicity of Ara-C may be enhanced by this ability to impact the action of topoisomerase I.Human topoisomerase I is 765 amino acids (91 kDa) and is composed of four domains: N-terminal (residues 1–200), core (201–635), linker (636–712), and C-terminal domain (713–765). Several crystal structures of human topoisomerase I DNA complexes have been determined (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar, 21Redinbo M.R. Champoux J.J. Hol W.G. Biochemistry. 2000; 39: 6832-6840Google Scholar). Core subdomains I and II form the 舠CAP舡 of human topoisomerase I that contacts one side of the DNA, while core subdomain III, the 舠CAT,舡 and the C-terminal domain contact the opposite side of the DNA. The CAP and CAT regions of the enzyme together wrap completely around the DNA duplex and position the active site residues within hydrogen bonding distance of the scissile DNA phosphate group. Four of five active site residues, Arg-488, Lys-532, Arg-590, and His-632, are located in core subdomain III, while the catalytic Tyr-723 resides in the C-terminal domain (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar).Structures of covalent human topoisomerase I-DNA complexes containing an intact 3′-phosphotyrosine linkage have also been reported (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). In these trapped covalent protein-DNA complexes, the scissile phosphate contained a bridging phosphorothiolate linkage, which, upon cleavage by topoisomerase I, generates a free 5′-sulfhydryl unable to participate in strand religation (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 22Burgin A.B. Methods Mol. Biol. 2001; 95: 119-128Google Scholar). The use of 5′-bridging phosphorothiolate linkages to trap covalent complexes has been successfully employed to examine several enzymes that form transient 3′-phosphotyrosine linkages, including eukaryotic type IB topoisomerases, viral topoisomerases, and bacterial and phage tyrosine recombinases and integrases (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 22Burgin A.B. Methods Mol. Biol. 2001; 95: 119-128Google Scholar, 23Burgin A. Huizenga B. Nash H. Nucleic Acids Res. 1995; 23: 2973-2979Google Scholar, 24Krogh B.O. Cheng C. Burgin A. Shuman S. Virology. 1999; 264: 441-451Google Scholar, 25Burgin A. Nash H. Curr. Biol. 1995; 5: 1312-1321Google Scholar, 26Hwang Y. Park M. Fischer W.H. Burgin A. Bushman F. Virology. 1999; 262: 479-491Google Scholar, 27Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Google Scholar, 28Kazmierczak R.A. Swalla B. Burgin A. Gumport R.I. Gardner J.F. Nucleic Acids Res. 2002; 30: 5193-5204Google Scholar). Detailed biochemical studies have shown that the presence of a 5′-bridging phosphorothiolate linkage has a marginal effect on the rate of cleavage by such enzymes (down ∼2-fold), but lowers the rate of religation by at least 10,000-fold (24Krogh B.O. Cheng C. Burgin A. Shuman S. Virology. 1999; 264: 441-451Google Scholar). In addition, x-ray crystallographic studies have revealed that when the active form of human topoisomerase I (with the Tyr-723 residue intact) is used for crystallization, a bridging phosphorothiolate linkage is required to obtain crystals (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar).We determined the 3.1 Å crystal structure of a human topoisomerase I in covalent complex with a 22-base pair oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand to elucidate the structural impact of Ara-C on this enzyme. This is only the third structure of a covalent topoisomerase I-DNA complex reported to date (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). We find that Ara-C introduces numerous subtle structural changes, including changes in sugar pucker and base position, that contribute to a new positioning of the free 5′-sulfhydryl away from the 3′-phosphotyrosine linkage. Thus, the single-strand religation reaction catalyzed by the enzyme is decreased, producing a longer lived covalent protein-DNA complex.DiscussionThe leukemia drug Ara-C contains a arabinose sugar ring rather than the ribose standard to DNA and RNA bases. As such, its 2′-hydroxyl group is oriented in a manner distinct from the equivalent RNA cytosine base (Fig. 1). Ara-C is thought to elicit its antineoplastic effects by acting as a competitive inhibitor of DNA polymerases α and ॆ (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar,17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 41Grant S. Front. Biosc. 1997; 2: 242-252Google Scholar). Even at low concentrations, however, the drug becomes incorporated into DNA and disrupts DNA metabolism (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar, 17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 41Grant S. Front. Biosc. 1997; 2: 242-252Google Scholar). Pourquier et al. (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar) have shown that the presence of an Ara-C base at the +1 position of the intact strand (opposite the site of single-strand cleavage) slows the rate of DNA strand religation by human topoisomerase I 2–3-fold (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). The extended lifetime of the covalent topoisomerase I-DNA complex may contribute to antineoplastic effects of Ara-C by enhancing chromosomal instability. Indeed, human leukemia cells that lack detectable levels of topoisomerase I are resistant to the effects of Ara-C (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar).We determined the 3.1 Å resolution crystal structure of human topoisomerase I in covalent complex with a 22-base pair DNA duplex containing Ara-C at the +1 position of the intact strand (Fig. 2). The structure reveals that the Ara-C non-standard 2′-hydroxyl introduces numerous subtle structural changes, particularly the +1 base pair (Fig.4A). The 2′-hydroxyl of Ara-C forms a hydrogen bond with the O4′ of the −1 sugar, which stabilizes the C3′-endo pucker exhibited by the arabinose ring of Ara-C (Fig. 4B). These structural changes cause the +1 base pair of the duplex to shift in position relative to the equivalent base pair in a covalent topoisomerase I DNA complex without a site of damage reported previously (1A31; Ref. 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). This, in turn, appears to cause the free 5′-sulfhydryl (which replaces the 5′-hydroxyl in this trapped covalent complex; 8, 19, 23–28) in the nicked DNA strand to shift away from the covalent phosphotyrosine linkage and form a hydrogen bond with the side chain of Asn-722, an interaction not observed in previous topoisomerase I covalent complexes (Figs. 4A and 6). Taken together, these results indicate that the subtle change of the duplex opposite the single-strand DNA break shifts the free 5′-end of the nicked strand away from the covalent 3′-phosphotyrosine linkage. These results likely explain the impact on topoisomerase I activity reported by Pourquier et al. (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar).This Ara-C structure provides additional insight into the catalytic mechanism of human topoisomerase I. As the active site residues are brought into place upon DNA binding, Asn-722 does not appear to contact the DNA, as observed in several non-covalent topoisomerase I DNA complexes (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar, 21Redinbo M.R. Champoux J.J. Hol W.G. Biochemistry. 2000; 39: 6832-6840Google Scholar). However, as the downstream region of DNA undergoes relaxation by the proposed controlled rotation mechanism, Asn-722 may have ample opportunity to hydrogen bond with the free 5′-hydroxyl of the nicked strand. Indeed, after relaxation slows, Asn-722 may play a crucial role via hydrogen bonding in guiding the 5′-hydroxyl into place for the religation phase of catalysis. This interaction is likely to be transitory in reactions involving non-damaged DNA. The change caused by the Ara-C base appears to stabilize this interaction, allowing us to visualize it in the structure presented here.The importance of Asn-722 in human topoisomerase I and the equivalent Asn-726 in S. cerevisiae topoisomerase I in the catalytic cycle and camptothecin sensitivity of the enzyme have been established by several careful biochemical studies. For example, mutation of Asn-722 to histidine in human topoisomerase I increases the rate of DNA cleavage, while mutation to aspartic acid decreases the DNA binding affinity of the enzyme (38Pourquier P. Pommier Y. Adv. Cancer Res. 2001; 80: 189-216Google Scholar). An N722S mutation in human topoisomerase I, in contrast, does not impact the catalytic activity of the enzyme but does reduce its sensitivity to camptothecin (40Fertala J. Vance J.R. Pourquier P. Pommier Y. Bjornsti M.A. J. Biol. Chem. 2000; 275: 15246-15253Google Scholar). We provide structural evidence in this and previous work that sites of DNA damage impact the ability of Asn-722 to align the active site of human topoisomerase I both before and after single-strand DNA cleavage by the enzyme (13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar). This residue may play a similar role with other DNA lesions that impact human topoisomerase I, including ethenoadenine adducts, wobble base pairs, and uracil mismatches. In summary, we show that relatively subtle modifications caused by the presence of a single 2′-hydroxyl group on the opposite side of the substrate DNA duplex can alter the structure of the human topoisomerase I active site and impact the catalytic action of the enzyme. Human topoisomerase I solves the DNA topological problems that arise from a wide variety of nuclear processes including replication, transcription, and recombination (1Champoux J.J. Annu. Rev. Biochem. 2001; 70: 369-413Google Scholar, 2Wang J.C. Annu. Rev. Biochem. 1996; 65: 635-692Google Scholar). The enzyme nicks one strand of duplex DNA using a transesterification reaction that produces a transient 3′-phosphotyrosine linkage and guides the relaxation of either positive or negative superhelical tension by a proposed 舠controlled rotation舡 mechanism (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar). The enzyme then catalyzes a second transesterification in which the free hydroxyl at the 5′-end of the nicked DNA strand attacks the phosphotyrosine bond, resealing the nick, and releasing a more relaxed DNA molecule. Topoisomerase I plays a vital role in maintaining DNA stability and is known to travel with active replication and transcription complexes in human cells (4Pommier Y. Pourquier P. Fan Y. Strumberg D. Biochim. Biophys. Acta. 1998; 1400: 83-105Google Scholar,5Holden J.A. Curr. Med. Chem. Anti-Cancer Agents. 2001; 1: 1-25Google Scholar). Human topoisomerase I is the sole target of the camptothecins (CPT), a potent class of anticancer drugs used to treat late-term solid malignancies (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 4Pommier Y. Pourquier P. Fan Y. Strumberg D. Biochim. Biophys. Acta. 1998; 1400: 83-105Google Scholar, 6Hsiang Y.H. Hertzberg R. Hecht S. Liu L.F. J. Biol. Chem. 1985; 260: 14873-14878Google Scholar). Camptothecin effectively targets the religation phase of topoisomerase I catalysis by stabilizing the covalent protein-DNA complex and trapping the enzyme on DNA (7Hertzberg R.P. Caranfa M.J. Hecht S.M. Biochemistry. 1989; 28: 4629-4638Google Scholar, 8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar). In this way, CPT converts topoisomerase I into a cellular poison. Human topoisomerase I is also affected by several forms of DNA damage, including abasic lesions, wobble base pairs, and base pair mismatches (9Pourquier P. Ueng L.M. Kohlhagen G. Mazumder A. Gupta M. Kohn K.W. Pommier Y. J. Biol. Chem. 1997; 272: 7792-7796Google Scholar, 10Pourquier P. Bjornsti M.A. Pommier Y. J. Biol. Chem. 1998; 273: 27245-27249Google Scholar, 11Pourquier P. Ueng L.M. Fertala J. Wang D. Park H.J. Essigmann J.M. Bjornsti M.A. Pommier Y. J. Biol. Chem. 1999; 274: 8516-8523Google Scholar, 12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar). Such lesions can impact each stage of topoisomerase I's catalytic cycle, including DNA binding, single-strand DNA cleavage, and religation. 1-ॆ-d-Arabinofuranosylcytosine (Ara-C)1 is a nucleoside analogue used in the treatment of acute leukemia (14Grant S. Adv. Cancer Res. 2002; 72: 197-233Google Scholar, 15Mastrianni D.M. Tung N.M. Tenen D.G. Am. J. Med. 1992; 92: 286-295Google Scholar). Ara-C and the standard cytosine DNA base differ by the presence of a 2′-hydroxyl on the arabinose ring of the drug (Fig.1). Ara-C is thought to inhibit DNA polymerases central to replication and repair processes, and thus to slow the growth of malignant cells (16Collins A.R.S. Biochim. Biophys. Acta. 1977; 478: 461-473Google Scholar, 17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar). The detailed impact of Ara-C on human cells, however, is poorly understood. Incorporation of Ara-C into DNA causes localized alterations in the DNA duplex, including changes in sugar pucker, base stacking, and backbone torsion angles (17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 18Gao Y.-G. van der Marel G.A. van Boom J.H. Wang A.H.J. Biochemistry. 1991; 30: 9922-9931Google Scholar). Biochemical studies using human topoisomerase I have revealed that Ara-C incorporation at the +1 position of the intact (non-scissile) strand adjacent to the site of single-strand DNA cleavage induces a 4–6-fold increase in covalent topoisomerase I-DNA complexes caused by a 2–3-fold decrease in the rate of religation by the enzyme (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). Because the stabilization of covalent topoisomerase I-DNA complexes converts the enzyme into a cellular poison, the cytotoxicity of Ara-C may be enhanced by this ability to impact the action of topoisomerase I. Human topoisomerase I is 765 amino acids (91 kDa) and is composed of four domains: N-terminal (residues 1–200), core (201–635), linker (636–712), and C-terminal domain (713–765). Several crystal structures of human topoisomerase I DNA complexes have been determined (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar, 21Redinbo M.R. Champoux J.J. Hol W.G. Biochemistry. 2000; 39: 6832-6840Google Scholar). Core subdomains I and II form the 舠CAP舡 of human topoisomerase I that contacts one side of the DNA, while core subdomain III, the 舠CAT,舡 and the C-terminal domain contact the opposite side of the DNA. The CAP and CAT regions of the enzyme together wrap completely around the DNA duplex and position the active site residues within hydrogen bonding distance of the scissile DNA phosphate group. Four of five active site residues, Arg-488, Lys-532, Arg-590, and His-632, are located in core subdomain III, while the catalytic Tyr-723 resides in the C-terminal domain (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar). Structures of covalent human topoisomerase I-DNA complexes containing an intact 3′-phosphotyrosine linkage have also been reported (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). In these trapped covalent protein-DNA complexes, the scissile phosphate contained a bridging phosphorothiolate linkage, which, upon cleavage by topoisomerase I, generates a free 5′-sulfhydryl unable to participate in strand religation (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 22Burgin A.B. Methods Mol. Biol. 2001; 95: 119-128Google Scholar). The use of 5′-bridging phosphorothiolate linkages to trap covalent complexes has been successfully employed to examine several enzymes that form transient 3′-phosphotyrosine linkages, including eukaryotic type IB topoisomerases, viral topoisomerases, and bacterial and phage tyrosine recombinases and integrases (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar, 22Burgin A.B. Methods Mol. Biol. 2001; 95: 119-128Google Scholar, 23Burgin A. Huizenga B. Nash H. Nucleic Acids Res. 1995; 23: 2973-2979Google Scholar, 24Krogh B.O. Cheng C. Burgin A. Shuman S. Virology. 1999; 264: 441-451Google Scholar, 25Burgin A. Nash H. Curr. Biol. 1995; 5: 1312-1321Google Scholar, 26Hwang Y. Park M. Fischer W.H. Burgin A. Bushman F. Virology. 1999; 262: 479-491Google Scholar, 27Krogh B.O. Shuman S. Mol. Cell. 2000; 5: 1035-1041Google Scholar, 28Kazmierczak R.A. Swalla B. Burgin A. Gumport R.I. Gardner J.F. Nucleic Acids Res. 2002; 30: 5193-5204Google Scholar). Detailed biochemical studies have shown that the presence of a 5′-bridging phosphorothiolate linkage has a marginal effect on the rate of cleavage by such enzymes (down ∼2-fold), but lowers the rate of religation by at least 10,000-fold (24Krogh B.O. Cheng C. Burgin A. Shuman S. Virology. 1999; 264: 441-451Google Scholar). In addition, x-ray crystallographic studies have revealed that when the active form of human topoisomerase I (with the Tyr-723 residue intact) is used for crystallization, a bridging phosphorothiolate linkage is required to obtain crystals (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). We determined the 3.1 Å crystal structure of a human topoisomerase I in covalent complex with a 22-base pair oligonucleotide containing Ara-C at the +1 position of the non-scissile DNA strand to elucidate the structural impact of Ara-C on this enzyme. This is only the third structure of a covalent topoisomerase I-DNA complex reported to date (8Staker B.L. Hjerrild K. Feese M.D. Behnke C.A. Burgin A.B. Stewart L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15387-15392Google Scholar, 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). We find that Ara-C introduces numerous subtle structural changes, including changes in sugar pucker and base position, that contribute to a new positioning of the free 5′-sulfhydryl away from the 3′-phosphotyrosine linkage. Thus, the single-strand religation reaction catalyzed by the enzyme is decreased, producing a longer lived covalent protein-DNA complex. DiscussionThe leukemia drug Ara-C contains a arabinose sugar ring rather than the ribose standard to DNA and RNA bases. As such, its 2′-hydroxyl group is oriented in a manner distinct from the equivalent RNA cytosine base (Fig. 1). Ara-C is thought to elicit its antineoplastic effects by acting as a competitive inhibitor of DNA polymerases α and ॆ (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar,17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 41Grant S. Front. Biosc. 1997; 2: 242-252Google Scholar). Even at low concentrations, however, the drug becomes incorporated into DNA and disrupts DNA metabolism (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar, 17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 41Grant S. Front. Biosc. 1997; 2: 242-252Google Scholar). Pourquier et al. (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar) have shown that the presence of an Ara-C base at the +1 position of the intact strand (opposite the site of single-strand cleavage) slows the rate of DNA strand religation by human topoisomerase I 2–3-fold (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). The extended lifetime of the covalent topoisomerase I-DNA complex may contribute to antineoplastic effects of Ara-C by enhancing chromosomal instability. Indeed, human leukemia cells that lack detectable levels of topoisomerase I are resistant to the effects of Ara-C (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar).We determined the 3.1 Å resolution crystal structure of human topoisomerase I in covalent complex with a 22-base pair DNA duplex containing Ara-C at the +1 position of the intact strand (Fig. 2). The structure reveals that the Ara-C non-standard 2′-hydroxyl introduces numerous subtle structural changes, particularly the +1 base pair (Fig.4A). The 2′-hydroxyl of Ara-C forms a hydrogen bond with the O4′ of the −1 sugar, which stabilizes the C3′-endo pucker exhibited by the arabinose ring of Ara-C (Fig. 4B). These structural changes cause the +1 base pair of the duplex to shift in position relative to the equivalent base pair in a covalent topoisomerase I DNA complex without a site of damage reported previously (1A31; Ref. 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). This, in turn, appears to cause the free 5′-sulfhydryl (which replaces the 5′-hydroxyl in this trapped covalent complex; 8, 19, 23–28) in the nicked DNA strand to shift away from the covalent phosphotyrosine linkage and form a hydrogen bond with the side chain of Asn-722, an interaction not observed in previous topoisomerase I covalent complexes (Figs. 4A and 6). Taken together, these results indicate that the subtle change of the duplex opposite the single-strand DNA break shifts the free 5′-end of the nicked strand away from the covalent 3′-phosphotyrosine linkage. These results likely explain the impact on topoisomerase I activity reported by Pourquier et al. (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar).This Ara-C structure provides additional insight into the catalytic mechanism of human topoisomerase I. As the active site residues are brought into place upon DNA binding, Asn-722 does not appear to contact the DNA, as observed in several non-covalent topoisomerase I DNA complexes (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar, 21Redinbo M.R. Champoux J.J. Hol W.G. Biochemistry. 2000; 39: 6832-6840Google Scholar). However, as the downstream region of DNA undergoes relaxation by the proposed controlled rotation mechanism, Asn-722 may have ample opportunity to hydrogen bond with the free 5′-hydroxyl of the nicked strand. Indeed, after relaxation slows, Asn-722 may play a crucial role via hydrogen bonding in guiding the 5′-hydroxyl into place for the religation phase of catalysis. This interaction is likely to be transitory in reactions involving non-damaged DNA. The change caused by the Ara-C base appears to stabilize this interaction, allowing us to visualize it in the structure presented here.The importance of Asn-722 in human topoisomerase I and the equivalent Asn-726 in S. cerevisiae topoisomerase I in the catalytic cycle and camptothecin sensitivity of the enzyme have been established by several careful biochemical studies. For example, mutation of Asn-722 to histidine in human topoisomerase I increases the rate of DNA cleavage, while mutation to aspartic acid decreases the DNA binding affinity of the enzyme (38Pourquier P. Pommier Y. Adv. Cancer Res. 2001; 80: 189-216Google Scholar). An N722S mutation in human topoisomerase I, in contrast, does not impact the catalytic activity of the enzyme but does reduce its sensitivity to camptothecin (40Fertala J. Vance J.R. Pourquier P. Pommier Y. Bjornsti M.A. J. Biol. Chem. 2000; 275: 15246-15253Google Scholar). We provide structural evidence in this and previous work that sites of DNA damage impact the ability of Asn-722 to align the active site of human topoisomerase I both before and after single-strand DNA cleavage by the enzyme (13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar). This residue may play a similar role with other DNA lesions that impact human topoisomerase I, including ethenoadenine adducts, wobble base pairs, and uracil mismatches. In summary, we show that relatively subtle modifications caused by the presence of a single 2′-hydroxyl group on the opposite side of the substrate DNA duplex can alter the structure of the human topoisomerase I active site and impact the catalytic action of the enzyme. The leukemia drug Ara-C contains a arabinose sugar ring rather than the ribose standard to DNA and RNA bases. As such, its 2′-hydroxyl group is oriented in a manner distinct from the equivalent RNA cytosine base (Fig. 1). Ara-C is thought to elicit its antineoplastic effects by acting as a competitive inhibitor of DNA polymerases α and ॆ (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar,17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 41Grant S. Front. Biosc. 1997; 2: 242-252Google Scholar). Even at low concentrations, however, the drug becomes incorporated into DNA and disrupts DNA metabolism (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar, 17Schweitzer B.I. Mikita T. Kellogg G.W. Gardner K.H. Beardsley G.P. Biochemistry. 1994; 33: 11460-11475Google Scholar, 41Grant S. Front. Biosc. 1997; 2: 242-252Google Scholar). Pourquier et al. (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar) have shown that the presence of an Ara-C base at the +1 position of the intact strand (opposite the site of single-strand cleavage) slows the rate of DNA strand religation by human topoisomerase I 2–3-fold (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). The extended lifetime of the covalent topoisomerase I-DNA complex may contribute to antineoplastic effects of Ara-C by enhancing chromosomal instability. Indeed, human leukemia cells that lack detectable levels of topoisomerase I are resistant to the effects of Ara-C (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). We determined the 3.1 Å resolution crystal structure of human topoisomerase I in covalent complex with a 22-base pair DNA duplex containing Ara-C at the +1 position of the intact strand (Fig. 2). The structure reveals that the Ara-C non-standard 2′-hydroxyl introduces numerous subtle structural changes, particularly the +1 base pair (Fig.4A). The 2′-hydroxyl of Ara-C forms a hydrogen bond with the O4′ of the −1 sugar, which stabilizes the C3′-endo pucker exhibited by the arabinose ring of Ara-C (Fig. 4B). These structural changes cause the +1 base pair of the duplex to shift in position relative to the equivalent base pair in a covalent topoisomerase I DNA complex without a site of damage reported previously (1A31; Ref. 19Redinbo M.R. Stewart L. Kuhn P. Champoux J.J. Hol W.G. Science. 1998; 279: 1504-1513Google Scholar). This, in turn, appears to cause the free 5′-sulfhydryl (which replaces the 5′-hydroxyl in this trapped covalent complex; 8, 19, 23–28) in the nicked DNA strand to shift away from the covalent phosphotyrosine linkage and form a hydrogen bond with the side chain of Asn-722, an interaction not observed in previous topoisomerase I covalent complexes (Figs. 4A and 6). Taken together, these results indicate that the subtle change of the duplex opposite the single-strand DNA break shifts the free 5′-end of the nicked strand away from the covalent 3′-phosphotyrosine linkage. These results likely explain the impact on topoisomerase I activity reported by Pourquier et al. (12Pourquier P. Takebayashi Y. Urasaki Y. Gioffre C. Kohlhagen G. Pommier Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1885-1890Google Scholar). This Ara-C structure provides additional insight into the catalytic mechanism of human topoisomerase I. As the active site residues are brought into place upon DNA binding, Asn-722 does not appear to contact the DNA, as observed in several non-covalent topoisomerase I DNA complexes (3Stewart L. Redinbo M.R. Qiu X. Hol W.G. Champoux J.J. Science. 1998; 279: 1534-1541Google Scholar, 13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar, 20Redinbo M.R. Stewart L. Champoux J.J. Hol W.G. J. Mol. Biol. 1999; 292: 685-696Google Scholar, 21Redinbo M.R. Champoux J.J. Hol W.G. Biochemistry. 2000; 39: 6832-6840Google Scholar). However, as the downstream region of DNA undergoes relaxation by the proposed controlled rotation mechanism, Asn-722 may have ample opportunity to hydrogen bond with the free 5′-hydroxyl of the nicked strand. Indeed, after relaxation slows, Asn-722 may play a crucial role via hydrogen bonding in guiding the 5′-hydroxyl into place for the religation phase of catalysis. This interaction is likely to be transitory in reactions involving non-damaged DNA. The change caused by the Ara-C base appears to stabilize this interaction, allowing us to visualize it in the structure presented here. The importance of Asn-722 in human topoisomerase I and the equivalent Asn-726 in S. cerevisiae topoisomerase I in the catalytic cycle and camptothecin sensitivity of the enzyme have been established by several careful biochemical studies. For example, mutation of Asn-722 to histidine in human topoisomerase I increases the rate of DNA cleavage, while mutation to aspartic acid decreases the DNA binding affinity of the enzyme (38Pourquier P. Pommier Y. Adv. Cancer Res. 2001; 80: 189-216Google Scholar). An N722S mutation in human topoisomerase I, in contrast, does not impact the catalytic activity of the enzyme but does reduce its sensitivity to camptothecin (40Fertala J. Vance J.R. Pourquier P. Pommier Y. Bjornsti M.A. J. Biol. Chem. 2000; 275: 15246-15253Google Scholar). We provide structural evidence in this and previous work that sites of DNA damage impact the ability of Asn-722 to align the active site of human topoisomerase I both before and after single-strand DNA cleavage by the enzyme (13Lesher D-T.T. Pommier Y. Stewart L. Redinbo M.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12102-12107Google Scholar). This residue may play a similar role with other DNA lesions that impact human topoisomerase I, including ethenoadenine adducts, wobble base pairs, and uracil mismatches. In summary, we show that relatively subtle modifications caused by the presence of a single 2′-hydroxyl group on the opposite side of the substrate DNA duplex can alter the structure of the human topoisomerase I active site and impact the catalytic action of the enzyme. We thank S. Bencharit, R. Watkins, and Y. Xue for thoughtful discussions and assistance in creating figures." @default.
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• W2052555812 title "Structural Impact of the Leukemia Drug 1-ॆ-d-Arabinofuranosylcytosine (Ara-C) on the Covalent Human Topoisomerase I-DNA Complex" @default.
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