Tryptophane-205 of human topoisomerase I is essential for camptothecin inhibition of negative but not positive supercoil removal

Positive supercoils are introduced in cellular DNA in front of and negative supercoils behind tracking polymerases. Since DNA purified from cells is normally under-wound, most studies addressing the relaxation activity of topoisomerase I have utilized negatively supercoiled plasmids. The present report compares the relaxation activity of human topoisomerase I variants on plasmids containing equal numbers of superhelical twists with opposite handedness. We demonstrate that the wild-type enzyme and mutants lacking amino acids 1–206 or 191–206, or having tryptophane-205 replaced with a glycine relax positive supercoils faster than negative supercoils under both processive and distributive conditions. In contrast to wild-type topoisomerase I, which exhibited camptothecin sensitivity during relaxation of both negative and positive supercoils, the investigated N-terminally mutated variants were sensitive to camptothecin only during removal of positive supercoils. These data suggest different mechanisms of action during removal of supercoils of opposite handedness and are consistent with a recently published simulation study [Sari and Andricioaei (2005) Nucleic Acids Res., 33, 6621–6634] suggesting flexibility in distinct parts of the enzyme during clockwise or counterclockwise strand rotation

Topoisomerase 1 inhibits MYC promoter activity by inducing G-quadruplex formation

We have investigated the function of human topoisomerase 1 (TOP1) in regulation of G-quadruplex (G4) formation in the Pu27 region of the MYC P1 promoter. Pu27 is among the best characterized G4 forming sequences in the human genome and it is well known that promoter activity is inhibited upon G4 formation in this region. We found that TOP1 downregulation stimulated transcription from a promoter with wildtype Pu27 but not if the G4 motif in Pu27 was interrupted by mutation(s). The effect was not specific to the MYC promoter and similar results were obtained for the G4 forming promoter element WT21. The other major DNA topoisomerases with relaxation activity, topoisomerases 2α and β, on the other hand, did not affect G4 dependent promoter activity. The cellular studies were supported by in vitro investigations demonstrating a high affinity of TOP1 for wildtype Pu27 but not for mutant sequences unable to form G4. Moreover, TOP1 was able to induce G4 formation in Pu27 inserted in double stranded plasmid DNA in vitro. This is the first time TOP1 has been demonstrated capable of inducing G4 formation in double stranded DNA and of influencing G4 formation in cells

Assembly and structural analysis of a covalently closed nano-scale DNA cage

The inherent properties of DNA as a stable polymer with unique affinity for partner molecules determined by the specific Watson–Crick base pairing makes it an ideal component in self-assembling structures. This has been exploited for decades in the design of a variety of artificial substrates for investigations of DNA-interacting enzymes. More recently, strategies for synthesis of more complex two-dimensional (2D) and 3D DNA structures have emerged. However, the building of such structures is still in progress and more experiences from different research groups and different fields of expertise are necessary before complex DNA structures can be routinely designed for the use in basal science and/or biotechnology. Here we present the design, construction and structural analysis of a covalently closed and stable 3D DNA structure with the connectivity of an octahedron, as defined by the double-stranded DNA helices that assembles from eight oligonucleotides with a yield of ∼30%. As demonstrated by Small Angle X-ray Scattering and cryo-Transmission Electron Microscopy analyses the eight-stranded DNA structure has a central cavity larger than the apertures in the surrounding DNA lattice and can be described as a nano-scale DNA cage, Hence, in theory it could hold proteins or other bio-molecules to enable their investigation in certain harmful environments or even allow their organization into higher order structures

Forskningslignende laboratorieaktiviteter for 1. års studerende på universitetet – muligheder og udfordringer

Forskningsbaseret undervisning er en hjørnesten i universitetsundervisning, herunder både arbejdet med forskningens indhold og forskningsmetode. I dette studie undersøges hvordan førsteårs studerende vurderer udbyttet fra arbejdet med autentisk forskning. Baseret på data fra gentaget spørgeskema på to førsteårskurser i henholdsvis bioteknologi og kemi identificeres positive udbytter knyttet til at møde forskere i et autentisk forskningsmiljø og til at indgå i forskningslignende aktiviteter med en vis autonomi. Der ses dog også tegn på positiv effekt af en tydelig rammesætning og feedback. Udfordringer er særligt knyttet til mødet med kompleks teori tidligt i studieforløbet. Resultaterne perspektiveres til laboratorieundervisning i gymnasieskole og læreruddannelse

Topoisomerase I as a Biomarker: Detection of Activity at the Single Molecule Level

Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response

Decreased camptothecin sensitivity of the stem-cell-like fraction of Caco2 cells correlates with an altered phosphorylation pattern of topoisomerase I.

The CD44+ and CD44- subpopulations of the colorectal cancer cell line Caco2 were analyzed separately for their sensitivities to the antitumor drug camptothecin. CD44+ cells were less sensitive to camptothecin than CD44- cells. The relative resistance of CD44+ cells was correlated with (i) reduced activity of the nuclear enzyme topoisomerase I and (ii) insensitivity of this enzyme to camptothecin when analyzed in extracts. In contrast, topoisomerase I activity was higher in extracts from CD44- cells and the enzyme was camptothecin sensitive. Topoisomerase I from the two subpopulations were differentially phosphorylated in a manner that appeared to determine the drug sensitivity and activity of the enzyme. This finding was further supported by the fact that phosphorylation of topoisomerase I in CD44+ cell extract by protein kinase CK2 converted the enzyme to a camptothecin sensitive, more active form mimicking topoisomerase I in extracts from CD44- cells. Conversely, dephosphorylation of topoisomerase I in extracts from CD44- cells rendered the enzyme less active and camptothecin resistant. These findings add to our understanding of chemotherapy resistance in the Caco2 CD44+ cancer stem cell model

Phoshorylation pattern of TopI in extracts from CD44+ or CD44− cell subpopulations.

<p>(<b>A</b>) Two-dimensional silver staining (left panel) and immunoblot analysis (right panel) of TopI expression pattern in the Caco2 cell line. The positions of TopI are underlined. The TopI identity of two major isoforms recognized by TopI antibodies was ensured by the MS/MS analysis of several bands excised from the silver stained gel (data not shown). (<b>B</b>) One-dimentional immunoblot analysis of TopI expression in CD44− and CD44+ FACS sorted cell fractions by immunoblotting with TopI antibodies. β-actin was used as a loading control. (<b>C</b>) Two-dimentional immunoblot analysis of TopI expression and post-translational modification pattern in CD44− and CD44+ cell fractions (left-hand panels) by immunoblotting with TopI antibodies. Equal protein amounts were loaded on the gels. The time of blot exposure is identical. The resulting pattern of TopI after cell treatment with λ-PPase prior the 2D PAGE analysis is presented (right-hand panels). Arrowheads indicate multiple forms of TopI (black arrowheads – the position of the non-phosphorylated TopI, blue arrowheads – the positions of phosphorylated TopI isoforms; red arrowheads – the positions of non-phosphorylated otherwise modified isoforms). The positions of parental and modified TopI isoforms are verified by the superimposing of all analyzed blots.</p