Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complexes with DNA

Redinbo, Matthew R.; Stewart, Lance; Kühn, Peter; Champoux, James J.; Hól, Wim G. J.

doi:10.1126/science.279.5356.1504

Cited by 834 publications

(912 citation statements)

References 54 publications

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“…In the closed clamp configuration found in the cocrystal structure, the two lobes are covalently joined through a continuous ␣-helical chain (␣8) on one side of the DNA molecule and contact each other in the ''lips'' region on the opposite side of the DNA through the formation of a salt bridge between loops that extend from each of the lobes (Fig. 1C) (6).…”

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confidence: 99%

“…Based on the crystal structure, topoisomerase I is a bi-lobed protein that clamps completely around duplex DNA through protein-DNA phosphate interactions (6). The core domain of the protein can be further divided into subdomains I, II, and III.…”

mentioning

confidence: 99%

“…Based on limited proteolysis studies and the crystal structure (6,7), the protein can be divided into four discrete domains. An N-terminal domain comprising approximately the first quarter of the protein is highly charged and poorly conserved.…”

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

Carey

Schultz

Sisson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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In cocrystal structures of human topoisomerase I and DNA, the enzyme is tightly clamped around the DNA helix. After cleavage and covalent attachment of the enzyme to the 3 end at the nick, DNA relaxation requires rotation of the DNA helix downstream of the cleavage site. Models based on the cocrystal structure reveal that there is insufficient space in the protein for such DNA rotation without some deformation of the cap and linker regions of the enzyme. Alternatively, it is conceivable that the protein clamp opens to facilitate the rotation process. To distinguish between these two possibilities, we engineered two cysteines into the opposing loops of the ''lips'' region of the enzyme, which allowed us to lock the protein via a disulfide crosslink in the closed conformation around the DNA. Importantly, the rate of DNA relaxation when the enzyme was locked on the DNA was comparable to that observed in the absence of the disulfide crosslink. These results indicate that DNA relaxation likely proceeds without extensive opening of the enzyme clamp. D NA topoisomerases are ubiquitous and essential enzymes that solve the topological problems accompanying key nuclear processes such as DNA replication, transcription, repair, and chromatin assembly by introducing temporary single-or double-strand breaks in the DNA (1-4). In addition, these enzymes fine-tune the steady-state level of DNA supercoiling to facilitate protein interactions with DNA and to prevent excessive supercoiling that is deleterious. There are two fundamental types of topoisomerases, which differ in both mechanism and cellular function (1). Type II enzymes are dimeric, promote the passage of one region of duplex DNA through a double-stranded break in the same or a different molecule, and are primarily dedicated to such processes as DNA supercoiling and chromosome segregation. Type I enzymes are monomeric and transiently break one strand of the duplex DNA, allowing for adjustments in helical winding. These enzymes are primarily responsible for removing torsional stress generated by processes that leave the DNA overwound or underwound. The type I topoisomerases have been further divided into two subfamilies based on sequence comparisons and reaction mechanisms (2). 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. The resulting formation of a phosphodiester bond between the tyrosine and the 3Ј end of the cleaved strand enables the enzyme to reseal the DNA by simple reversal of the cleavage reaction (1).Human topoisomerase I is a 765...

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

Carey

Schultz

Sisson

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Nup98-top1 Fusion Exhibits Both Overlapping and Distinct Promentioning

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

Gurevich

Aplan

Humphries

2004

Blood

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Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies, resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98, and more than one half of these are homeobox transcription factors. By contrast, the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1), a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors, evidenced by a competitive growth advantage in liquid culture, increased replating efficient of colony-forming cells (CFCs), and a marked increase in spleen colonyforming cell output. Moreover, in a murine bone marrow transplantation model, NUP98-TOP1 expression led to a lethal, transplantable leukemia characterized by extremely high white cell counts, splenomegaly, and mild anemia. Strikingly, a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings, together with similar biologic effects reported for NUP98-HOX fusions, suggest unexpected, overlapping functions of NUP98 fusion genes, perhaps related to common DNA binding properties. IntroductionA heterogenous group of hematopoietic malignancies has been described that are characterized by chromosomal translocations that create fusion genes involving the N-terminal portion of the nucleoporin gene, NUP98, on chromosome 11p15. 1 To date, 15 distinct fusion partners have been identified in NUP98 translocations. The most frequently observed fusion partners for NUP98 belong to the homeobox family of transcription factors and include HOXA9, 2,3 HOXD13, 4 HOXD11, 5 HOXA11, 6 HOXA13, 7,8 HOXC11,9 and HOXC13 10 as well as the nonclustered homeobox gene PMX1. 11 Recent studies in murine model systems have clearly demonstrated that both NUP98-HOXA9 and NUP98-HOXD13 play a causal and overt role in the pathogenesis of leukemia. 12,13 Furthermore, the disease onset can be greatly accelerated in these models by coexpression of the NUP98-HOX fusion gene and the HOX co-factor Meis1. These reports are consistent with the observations that HOX genes play key roles in the regulation of hematopoiesis and that overexpression of select HOX genes (eg, HOXA10, 14 HOXB3 15 ) lead to leukemia.In addition to the HOX genes, 7 "variant" partner genes that are associated with a wide range of biologic functions have been identified in fusion with NUP98 in hematopoietic malignancies. These include DDX10, 16,17 RAP1GDS1,18,19 Topoisomerase I, 20 LEDGF, 21 NSD1, 22 NSD3, 23 and Adducin 3. 24 These NUP98 fusion partner genes are diverse in function and, in contrast to the HOX fusion partners, are not known to have a specific or unique function in hematopoiesis.An intriguing example of one such non-HOX partner is Topoisomerase I (...

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A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps

et al. 1999

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A Monte Carlo procedure, encoded in the program Blob, has been developed and tested for the purpose of positioning large molecular fragments or small flexible molecules in electron density maps. The search performed by the algorithm appears to be sufficiently thorough to accurately position a small flexible ligand in well-defined density while remaining sufficiently random to offer interesting alternate suggestions for density representing disordered binding modes of a ligand. Furthermore, the algorithm is shown to be efficient enough to accurately position large rigid molecular fragments. In the first of the test cases with large molecular fragments, Blob was surprisingly effective in positioning a poly-alanine model of a 53-residue domain in poor electron density resulting from molecular replacement with a partial model. At 3.0 A resolution the domain was positioned consistently within 0.2 A of its experimentally determined position. Even at 6.0 A resolution Blob could consistently position the domain to within 0.75 A of its actual position. A second set of tests with large molecular fragments revealed that Blob could correctly position large molecular fragments with quite significant deviations from the actual structure. In this test case, fragments ranging from a 170-residue protein domain with a 3.8 A rms deviation from the actual structure to a 22-base pair ideal B-form DNA duplex were positioned accurately in a 3.2 A electron density map derived from multiple isomorphous replacement methods. Even when decreasing the quality of the maps, from a figure of merit of 0.57 to as low as 0. 35, Blob could still effectively position the large protein domain and the DNA duplex. Since it is efficient, can handle large molecular fragments, and works in poor and low resolution maps, Blob could be a useful tool for interpreting electron density maps in de novo structure determinations and in molecular replacement studies. Proteins 1999;36:512-525.

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Crystal Structures of Human Topoisomerase I in Covalent and Noncovalent Complexes with DNA

Cited by 834 publications

References 54 publications

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

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

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

A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps

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