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Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs.
No full textThe budding yeast Saccharomyces cerevisiae has been exploited to investigate the cytotoxic mechanisms of drugs that target DNA topoisomerases. This model organism has been used to establish eukaryotic DNA topoisomerase I or II as the cellular target of specific antineoplastic agents, to define mutations in these enzymes that confer drug resistance and to elucidate the cellular factors that modulate cell sensitivity to DNA topoisomerase-targeted drugs. These findings have provided valuable insights into the critical activities of these enzymes and how perturbing their functions produces DNA damage and cell death
Visualization of DNA Topoisomerases by Electron Microscopy.
No full textIn N. Bjornsti M-A and Osheroff (ed.
Study of Yeast DNA Topoisomerase II and Its Truncation Derivatives by Transmission Electron Microscopy
No full textThe 1429-amino acid residue long yeast DNA topoisomerase II and three of its deletion derivatives, a C-terminal truncation containing residues 1-1202, a 92-kDa fragment spanning residues 410-1202, and an A'-fragment spanning residues 660-1202, were examined by transmission electron microscopy. Analysis of rotary-shadowed images of these molecules shows that the full-length enzyme assumes a tripartite structure, in which a large globular core comprising the carboxyl parts of the dimeric enzyme is connected to a pair of smaller spherical masses comprising the ATPase domains of the enzyme. The linkers bridging the large globular structure and each of the smaller spheres are not visible in most of the images but appear to be sufficiently stiff to keep the relative positions of the connected parts. The angle extended by the pair of spherical masses is variable and falls in a range of 50-100 degrees for the majority of the images. On binding of a nonhydrolyzable ATP analog to the enzyme, this angle is significantly reduced as the two spherical masses swing into contact. These observations, together with results from previous biochemical and x-ray crystallographic studies of the enzyme, provide a sketch of the molecular architecture and conformational states of a catalytically active type II DNA topoisomerase
Protein concerted motions in the DNA-Human Topoisomerase I complex
No full textThe collective motions of the core and C-terminal domains of human topoisomerase I (topo I) have been analysed by molecular dynamics simulation of the protein in covalent complex with a 22 bp DNA duplex. The analysis evidenced a great number of correlated movements of core subdomain I and II residues, and a central role for helix 5 in the protein-DNA communication, in particular with the scissile strand downstream of the cleavage site. The flow of information between these core subdomains and DNA suggests that subdomains I and II play an essential role in the DNA relaxation process. In core subdomain III the majority of DNA contacting residues do not communicate with protein regions far from DNA, suggesting that they have a structural role. However, selected core subdomain III residues, involved in the orientation of the active site region, show correlated movements with residues distant from DNA, indicating that the information concerning the catalytic event is also transmitted. The flexibility of two loops formed by residues 519-520 and 580-584 seems indispensable to the dynamic participation of core subdomain III to the DNA cleavage and religation steps. The motion of specific residues has also been found to explain the effect of single point mutations that make topo I resistant to the anticancer drug camptothecin
Biologia Molecolare terza edizione
No full textLibro di testo per i corsi di biologia molecolare per le lauree triennali e specialistich
Template Supercoiling by a Yeast GAL4 Protein-Phage T7 RNA Polymerase Chimera. Science (1990) 249: 1261-1265
No full textFusion of the DNA-binding domain of yeast GAL4 protein to the amino terminus of bacteriophage T7 RNA polymerase yields a chimera that retains the characteristics of its components. The presence of the GAL4 peptide allows the chimeric enzyme to anchor itself on the DNA template, and this anchoring in turn drives the formation of a supercoiled DNA loop, in linear or circular templates, when RNA synthesis at the polymerase site forces a translocation of the DNA relative to the site. Nonspecific interaction between the chimeric enzyme and DNA appears to be sufficient to effect supercoiling during transcription. Transcription by the chimeric polymerase is strictly dependent on the presence of a T7 promoter; thus it provides a tool in vitro and in vivo for specifically supercoiling DNA segments containing T7 promoter sequences
Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine.
No full textA full-length human DNA topoisomerase I complementary DNA clone was mutagenized in vitro and the mutagenized DNA was used to replace wild-type human TOP1 complementary DNA in YCpGAL1-hTOP1, a plasmid constructed for the expression of the human enzyme in yeast. A yeast strain devoid of yeast DNA topoisomerase I and permeable to the anticancer drug camptothecin was transformed with the plasmid pool. Assays of DNA topoisomerase I in lysates of camptothecin-resistant transformants identified one with nearly the same level of the enzyme as transformants of unmutagenized YCpGAL1-hTOP1, and a single mutation changing Gly363 to a cysteine was found in this mutant. The G363C mutant enzyme was overexpressed in yeast and partially purified. It differed significantly from wild-type human DNA topoisomerase I similarly expressed and purified: camptothecin-stimulated cleavage of DNA was observed with the wild-type but not the G363C enzyme, and the DNA relaxation activity of the mutant enzyme, unlike that of the wild-type enzyme, was not significantly stimulated by Mg(II). The positions of the G363C and other previously reported camptothecin resistance mutations in eukaryotic DNA topoisomerase I were discussed in terms of a model in which the active site is an interdomainal cleft
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