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

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.Human topoisomerase I solves the DNA topological problems that arise from a wide variety of nuclear processes including replication, transcription, and recombination (1, 2). The enzyme nicks one strand of duplex DNA using a transesterification reaction that produces a transient 3Ј-phosphotyrosine linkage and guides the relaxation of either positive or negative superhelical tension by a proposed "controlled rotation" mechanism (3). The enzyme then catalyzes a second transesterification in which the free hydroxyl at the 5Ј-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 (4,5).Human topoisomerase I is the sole target of the camptothecins (CPT), a potent class of anticancer drugs used to treat late-term solid malignancies (3,4,6). Camptothecin effectively targets the religation phase of topoisomerase I catalysis by stabilizing the covalent protein-DNA complex and trapping the enzyme on DNA (7,8). 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 (9 -13). Such lesions can impact each stage of topoisomerase I's catalytic cycle, including DNA binding, single-strand DNA cleavage, and religation.

Rad9 Protects Cells from Topoisomerase Poison-induced Cell Death

“…The fact that cytarabine also requires Rad9 to induce S-phase arrest is consistent with earlier reports showing that cytarabine induces replication stress after its incorporation into DNA (66 -69). On the other hand, recent studies have suggested that cytarabine might act like a topoisomerase I poison (37,38). Evidence to support this claim has included (i) the demonstration that the religation step of topoisomerase I is slowed ϳ5-fold when an oligonucleotide containing cytidine arabinoside in place of cytidine at a topoisomerase I cleavage site is exposed to purified enzyme in vitro; (ii) the observation that covalent topoisomerase I-DNA complexes are increased in neoplastic cell lines several hours after the addition of cytarabine to cultures; and (iii) the demonstration that camptothecin-selected P388/camptothecin (CPT) murine lymphoma cells, which lack detectable topoisomerase I and are 4000-fold more resistant to CPT, are 5-fold resistant to cytarabine.…”

“…Aliquots derived from equal amounts of protein as assayed by the method of Bradford (46) were separated by SDS-10% PAGE. After polypeptides were transferred to Immobilon-P membranes (Millipore) and subjected to immunoblotting as described (38), bound antibodies were visualized by chemiluminescence (SuperSignal, Pierce).…”

“…Camptothecin, the founding member of a large class of topoisomerase I poisons (26,28), inhibits the religation step of topoisomerase I (29) to produce covalent topoisomerase I-DNA complexes (30,31) that likewise yield DNA double-strand breaks (32)(33)(34) after interaction with advancing replication forks (33,35). More recently, it has been reported that cytarabine, a nucleoside analogue that is widely used to treat acute leukemias and lymphomas (36), might also act by trapping topoisomerase I-DNA covalent complexes after incorporation of the analogue into DNA (37,38). Direct or indirect evidence has indicated that each of these prototypic anticancer agents also causes activation of Chk1 (19 -21, 39), which ordinarily occurs in an ATR-dependent manner (39,40).…”

Mechanisms of apoptosis induction by nucleoside analogs

“…Although the exact nature of the damage caused by nucleoside analogs in stalling replication forks is poorly understood, it follows that cellular responses to these agents are likely to overlap those elicited by drugs such as hydroxyurea (HU) that also cause replication forks to stall by limiting dNTP production. Also, crystallographic studies have demonstrated that incorporation of ara-C into DNA causes localized alterations in the DNA duplex (Schweitzer et al, 1994) and stabilizes covalent topoisomerase I-DNA complexes, thus converting the enzyme into a cellular poison (Pourquier et al, 2000;Chrencik et al, 2003). This suggests that cellular responses to nucleoside analogs are also likely to overlap those elicited by the camptothecin family of topoisomerase I active agents.…”

Substitution of Conserved Residues within the Active Site Alters the Cleavage Religation Equilibrium of DNA Topoisomerase I

“…23). The structural distortions induced by incorporation of Ara-C at the ϩ1 position of the complementary nonscissile strand also stabilize the covalent enzyme-DNA intermediate by reducing DNA religation (24). Mutation of conserved residues N-terminal to the active site tyrosine in the yeast and human enzymes also induces Top1p poisoning (12,(25)(26)(27).…”