The Domain Organization of Human Topoisomerase I

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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...

Section: Methodsmentioning

confidence: 99%

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

Carey

Schultz

Sisson

et al. 2003

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“…Human topoisomerase I breaks one strand of duplex DNA and relaxes superhelical tension by a postulated ''controlled rotation'' mechanism (6). The 765-aa enzyme is composed of four major regions: a highly charged and putatively unstructured N-terminal domain (residues 1-200; removed for structural studies), a conserved core domain (residues 201-635), a flexible linker domain (residues 636-712), and a conserved C-terminal domain (residues 713-765) that contains the catalytic Tyr-723 residue (7). Several crystal structures of human topoisomerase I in both covalent and noncovalent complexes with DNA have been reported (6,(8)(9)(10).…”

mentioning

confidence: 99%

8-Oxoguanine rearranges the active site of human topoisomerase I

Lesher

Pommier

Stewart

et al. 2002

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7,8-Dihydro-8-oxoguanine (8-oxoG) is the most common form of oxidative DNA damage in human cells. Biochemical studies have shown that 8-oxoG decreases the DNA cleavage activity of human topoisomerase I, an enzyme vital to DNA metabolism and stability. We present the 3.1-Å crystal structure of human topoisomerase I in noncovalent complex with a DNA oligonucleotide containing 8-oxoG at the ؉1 position in the scissile strand. We find that 8-oxoG reorganizes the active site of human topoisomerase I into an inactive conformation relative to the structures of topoisomerase I-DNA complexes elucidated previously. The catalytic Tyr-723-Phe rotates away from the DNA cleavage site and packs into the body of the molecule. A second active-site residue, Arg-590, becomes disordered and is not observed in the structure. The docked, inactive conformation of Tyr-723-Phe is reminiscent of the related tyrosine recombinase family of integrases and recombinases, suggesting a common regulatory mechanism. We propose that human topoisomerase I binds to DNA first in an inactive conformation and then rearranges its active site for catalysis. 8-OxoG appears to impact topoisomerase I by stabilizing the inactive, DNA-bound state.

“…This is unlikely to be in terms of catalytic efficiency. Separation of the core and catalytic domains of human topoisomerase IB by proteolytic cleavage within the unconserved linker does not markedly affect catalysis (32). Separate domains may permit the subunits to function independently, perhaps in conjunction with other partners (a possibility that we should explore with RNA interference studies).…”

Section: Discussionmentioning

confidence: 99%

An unusual type IB topoisomerase from African trypanosomes

Bodley

Chakraborty

Xie

et al. 2003

African trypanosomes are ancient eukaryotes that cause lethal disease in humans and cattle. Available drugs are inadequate and the need for new therapeutic targets is great. Trypanosoma brucei and related pathogens differ strikingly from higher eukaryotes in many aspects of nucleic acid structure and metabolism. We find yet another example of this in their unusual DNA topoisomerase IB. Type IB topoisomerases relieve the supercoils that accumulate during DNA and RNA synthesis, and are of considerable importance as the target for antitumor camptothecins. Dozens of type IB topoisomerases sequenced from eukaryotes, bacteria, and pox viruses are all encoded by a single gene that predictably contains a highly conserved DNA binding domain and C-terminal catalytic domain, linked by a nonconserved hydrophilic region. We find that topoisomerase IB in T. brucei is encoded by two genes: one for the DNA-binding domain and a second for the C-terminal catalytic domain. In keeping with this, highly purified fractions of native T. brucei topoisomerase IB catalytic activity contain two proteins, of 90 and 36 kDa. The native enzyme is conventional in its Mg 2؉ -independence, ability to relax positive and negative supercoils, and inhibition by camptothecin. Camptothecin promotes the formation of a covalent complex between 32 P-labeled substrate DNA and the small subunit. This unusual structural organization may provide a missing link in the evolution of type IB enzymes, which are thought to have arisen over time from the fusion of two independent domains. It also provides another basis for the design of selectively toxic drug candidates.T he African trypanosome, Trypanosoma brucei, is a flagellated protozoan that causes sleeping sickness in humans. This tsetse fly-transmitted meningoencephalitis is of increasing incidence and is fatal if not treated (1). Closely related organisms cause Chagas' disease and leishmaniasis. Available therapies for sleeping sickness are widely acknowledged to be inadequate: they require multiple parenteral doses for cure, are expensive, toxic, and are losing efficacy against drug-resistant parasites. Trypanosomes and leishmania are ancient eukaryotes whose distinctive features include structurally and metabolically unusual DNAs. In African trypanosomes, the nuclear genome is comprised of 11 chromosome pairs, several additional chromosomes of unknown ploidy, and Ϸ100 minichromosomes, each containing a single gene that encodes a variable surface protein (2). Surface protein genes can be activated by recombinationbased transfer from minichromosomes to transcriptionally active sites in larger chromosomes. Large polycistronic transcripts are trans-spliced to form mRNAs with a 5Ј-leader sequence and 3Ј poly(A) tail. Even more unusual is their mitochondrial DNA, termed kinetoplast or kDNA, which is in the form of a single gigantic network of interlocked relaxed DNA circles (3, 4). Mature mitochondrial messages are created by an editing process that involves systematic insertion or removal of U residues.DNA top...