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Volumn 17, Issue 4, 2007, Pages 1013-1017

Binding mode and affinity studies of DNA-binding agents using topoisomerase I DNA unwinding assay

Author keywords

DNA binding; Topoisomerase I unwinding assay

Indexed keywords

DNA TOPOISOMERASE; NAPHTHALENE DERIVATIVE;

EID: 33846604114     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2006.11.038     Document Type: Article
Times cited : (21)

References (33)
  • 11
    • 0001743059 scopus 로고    scopus 로고
    • DNA Intercalators
    • Kool E.T. (Ed), Elsevier, New York
    • Wilson W.D. DNA Intercalators. In: Kool E.T. (Ed). DNA and Aspects of Molecular Biology Vol. 7 (1999), Elsevier, New York 427
    • (1999) DNA and Aspects of Molecular Biology , vol.7 , pp. 427
    • Wilson, W.D.1
  • 14
    • 33846583827 scopus 로고    scopus 로고
    • note
    • Typically, 0.24 μg of supercoiled pUC19 plasmid DNA (Bayou Biolabs, Harahan, LA) was incubated with 5 U of human topoisomerase I enzyme (TopoGEN Inc., Port Orange, FL) for 5 min at 37 °C in 1× Topo I reaction buffer. The appropriate amount of compound was then added and the reaction mixture incubated for a further 1 hr at 37 °C. The reaction was terminated using 0.5% SDS and 0.5 mg/mL proteinase K. Subsequent incubation for an additional 15 min was followed by enzyme and compound extraction using a mixture of phenol/chloroform/isoamyl alcohol (25:24:1). The remaining DNA sample was then run on an agarose gel (1%) at 75 V for 3 h, stained with ethidium bromide, and photographed (type 667 film).
  • 15
    • 33846609831 scopus 로고    scopus 로고
    • note
    • -6 M in bp, 1.4 mL) over 24 s at 300 s intervals using a 250 μL syringe rotating at 290 rpm. Samples were degassed at 20 °C using a ThermoVac apparatus (MicroCal) before use. Each peak corresponded to the decrease in the power supplied to keep the temperatures of the sample and reference cells the same for each injection and represents the heat given off. In each case, response signals were corrected for the small heat of dilution associated with titrating the drug into the buffer. The heat of dilution for titrating buffer into DNA was found to be negligible. The heat released on binding was directly proportional to the amount of binding. A binding isotherm of heat released versus the molar ratio was constructed and the data fitted by non-linear least square fitting analysis to a model based on a single set of identical binding sites.
  • 16
    • 33846644699 scopus 로고    scopus 로고
    • note
    • 33
  • 17
    • 33846642845 scopus 로고    scopus 로고
    • note
    • m) studies were conducted on a Varian Cary 100 Bio UV-visible spectrophotometer attached to a temperature controller. Experiments were performed in 20 mM sodium phosphate buffer, pH 7.0. Melting profiles were obtained by measuring the absorbance at 260 nm as a function of temperature (25-95 °C) at a ramp rate of 0.5 °C/min. The concentration of DNA (calf thymus) was generally 10 μM (bp) with a DNA/drug ratio of 3:1.
  • 21
    • 33846605779 scopus 로고    scopus 로고
    • note
    • Experimental partition coefficient (log P) for each NDI compound was determined by the Shake-flask method by preparing a saturated solution in water, followed by thorough mixing with an equal volume of n-octanol. The mixture was then allowed to partition and equilibrate overnight. Subsequent spectrophotometric determination of the relative NDI concentration in n-octanol versus water layers yielded the n-octanol/water log P.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.