A Model for the Mechanism of Human Topoisomerase I

Abstract

The three-dimensional structure of a 70-kilodalton amino terminally truncated form of human topoisomerase I in complex with a 22-base pair duplex oligonucleotide, determined to a resolution of 2.8 angstroms, reveals all of the structural elements of the enzyme that contact DNA. The linker region that connects the central core of the enzyme to the carboxyl-terminal domain assumes a coiled-coil configuration and protrudes away from the remainder of the enzyme. The positively charged DNA-proximal surface of the linker makes only a few contacts with the DNA downstream of the cleavage site. In combination with the crystal structures of the reconstituted human topoisomerase I before and after DNA cleavage, this information suggests which amino acid residues are involved in catalyzing phosphodiester bond breakage and religation. The structures also lead to the proposal that the topoisomerization step occurs by a mechanism termed "controlled rotation."

DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein

“…The type IA subfamily is characterized by covalent attachment of the enzyme to the 5Ј end of the broken strand in the nicked intermediate, whereas all members of the type IB subfamily attach to the 3Ј end of the broken strand. The prototype of the type IB subfamily, human topoisomerase I, catalyzes changes in the superhelical state of duplex DNA by transiently breaking one strand of the DNA to allow rotation of one region of the duplex relative to another region (5). Strand cleavage is achieved by the nucleophilic attack of the active site tyrosine on a DNA phosphodiester bond.…”

“…One of the conclusions drawn from the crystal structure was that the available space within the protein framework downstream of the cleavage site is insufficient to easily accommodate the rotation of the DNA helix required for DNA relaxation (5). It was noted that the DNA proximal surfaces of the cap region and the linker contain 16 conserved positively charged side chains that could interact with the DNA and possibly hinder the rotation process.…”

NUP98-Topoisomerase I acute myeloid leukemia-associated fusion gene has potent leukemogenic activities independent of an engineered catalytic site mutation

“…Intriguingly, many of the non-HOX NUP98 fusion partners also contain domains predicted to adopt a coiled-coil conformation, 62 and this domain has been verified for TOP1 by x-ray crystallography. 52,53 Whether this domain promotes dimerization or is required for leukemic transformation, as is observed for several mixed-lineage leukemia (MLL) fusions, 63 awaits further investigation. Also intriguing is the observation of PB and BM from NUP98-TOP1 leukemic mice that exhibit high proportion of B220 lo cells.…”

“…13 TOP1 binds to DNA like a clamp with portions of the core domain and C-terminal domain, forming the upper and lower halves, respectively. 52,53 To ascertain whether these DNA binding domains are necessary for NUP98-TOP1 transformation, mutant constructs were engineered that deleted either the TOP1 core domain (NT ⌬CD) or the C-terminal domain (NT⌬C-term). Both mutants lost their in vitro growth-promoting activity as assayed in competitive liquid culture assays ( Figure 6C-D) or spleen colonyforming cell expansion assays (data not shown).…”

Phase I study of cisplatin, irinotecan, and epirubicin administered every 3 weeks in patients with advanced solid tumours

“…DNA topoisomerases I and II are nuclear enzymes that regulate the torsional strain of supercoiled DNA double helix during critical cellular processes such as replication, transcription, recombination, and repair (Stewart et al, 1998;Li and Liu, 2001;Topcu, 2001). Several antineoplastic agents have been found to exert their cytotoxic effects by inhibiting topoisomerases.…”