Journal:

Article Title: Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove

doi:

Figure Lengend Snippet: Et743 induces top1-mediated DNA single-strand breaks. SV40 DNA was reacted with top1 in the absence or presence of drugs, as indicated. Reactions were stopped with 0.5% SDS followed by proteinase K incubation. Samples were run in 1% agarose gels containing ethidium bromide. Percent nicked DNA was computed after FluorImager analysis (Molecular Dynamics) by using the imagequant software and is indicated below each lane. (A) top1-mediated nicking assay by using supercoiled negative SV40 DNA. Lane 1, DNA alone; lane 2, + top1; and lanes 3–6, + top1 and the indicated concentrations of Et743 or CPT. (B) Et743 induces more top1-mediated DNA nicking in supercoiled than in linear SV40 DNA. Lanes 1 and 5, DNA alone; lanes 2 and 6, DNA + top1; lanes 3 and 7, DNA + top1 + Et743 (0.1 μM); lanes 4 and 8, Et743 without top1. (C) Et743 does not induce detectable top2 cleavage complexes. Lane 1, DNA alone; lane 2, + top2; and lanes 3–4, + top2 and the indicated drug concentrations.

Article Snippet: Each reaction (10 μl final volume) in 10 mM Tris⋅HCl, pH 7.5/50 mM KCl/5 mM MgCl 2 /0.1 mM EDTA/1 mM ATP/15 μg/ml BSA contained 0.3 μg supercoiled simian virus 40 (SV40) DNA (GIBCO/BRL).

Techniques: Incubation, Software

Journal:

Article Title: Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove

doi:

Figure Lengend Snippet: DNA alkylation by Et743 induces salt-reversible top1-mediated DNA breaks at different sites from CPT. (A) Differences in sequence selectivity of top1-mediated DNA breaks for Et743 and CPT. The long BanI-HpaII fragments of SV40 DNA 3′-end-labeled at the BanI Site was used. Lane 1, formic acid (FA); lane 2, DNA alone; lane 3, + top1 without drug; lane 4, top1 + Et743 (10 μM); lane 5, top1 + CPT (10 μM). (B) Salt reversibility of the top1-mediated DNA breaks induced by Et743. Drug-induced DNA cleavage was reversed by adding 0.35 M NaCl (final concentration) (at time 0 corresponding to a 30-min incubation of top1 with Et743 or CPT; lanes 3 and 8, respectively) and by further incubation at 25°C for the indicated times. (C) Et743-DNA adducts induce the top1 cleavage complexes. 3′-end-labeled PvuII/HindIII fragment of pSK(−) phagemid DNA was used for these reactions. In lanes 2–9, reactions were performed in two consecutive steps. First, the DNA was treated overnight (O/N ≈16 hr) with or without drug, as indicated above pairs of lanes, then DNA was ethanol precipitated to remove free drug. Secondly, the DNA was reacted ± top1 in the absence of added drug (lanes 2–10) or in the presence of 10 μM CPT (lane 11). Lane 1, FA-sequencing lane.

Article Snippet: Each reaction (10 μl final volume) in 10 mM Tris⋅HCl, pH 7.5/50 mM KCl/5 mM MgCl 2 /0.1 mM EDTA/1 mM ATP/15 μg/ml BSA contained 0.3 μg supercoiled simian virus 40 (SV40) DNA (GIBCO/BRL).

Techniques: Sequencing, Labeling, Concentration Assay, Incubation