An unusual type IB topoisomerase from African trypanosomes

Abstract

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...

Effect of topoisomerase inhibitors and DNA-binding drugs on the cell proliferation and ultrastructure of Trypanosoma cruzi

“…Topoisomerases I and II have been characterized in several trypanosomatids [11][12][13][14][15][16][17][18][19]. Topo I activity is ATP-independent and the enzyme is composed of two subunits encoded by two different genes, with the C-terminal catalytic domain being highly conserved [17].…”

“…Topo I activity is ATP-independent and the enzyme is composed of two subunits encoded by two different genes, with the C-terminal catalytic domain being highly conserved [17]. Topo II has ATP-dependent and -independent decatenating activities and presents separate nuclear and mitochondrialencoding genes [20,21].…”

DNA topoisomerases in apicomplexan parasites: promising targets for drug discovery

“…DNA topoisomerases in Apicomplexa C. García-Estrada et al 1779(D'Arpa et al 1988. This four-domain modular structure is fairly well conserved in Eukarya with the notable exception of Trypanosomatids (trypanosomes and leishmanias), where TopIB is a heterodimeric enzyme consisting of a large subunit closely homologous to the core domain of other TopIB, and a small subunit includes the C-terminal domain containing the tyrosil residue that plays a role in DNA cleavage (Bodley et al 2003;Villa et al 2003).…”

N-terminal Region of the Large Subunit of Leishmania donovani Bisubunit Topoisomerase I Is Involved in DNA Relaxation and Interaction with the Smaller Subunit

“…Recently, the emergence of the bisubunit topoisomerase I of Trypanosoma (6) and Leishmania (7) in the kinetoplastid family have brought a new twist in topoisomerase research related to evolution and functional conservation of the type IB family. The core DNA binding domain and the catalytic domain harboring the consensus SKXXY motif lies in separate subunits.…”