Ethidium etd n i n pt n i sch 2 chph ptterpyhet ptts

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ethidium (Etd) ~ N ~ I - N-Pt-N /; - I ~ SCH 2 CHpH [Pt(terpy)(HET)]+, PtTS Figure 9,11 Organic (top) and inorganic (bottom) intercalators. 1 o 3.4A T Figure 9.12 Schematic representation of double-stranded DNA without (left) and with (shaded area, right) a bound intercalator (reproduced with permission from Reference 51). 1 o 10.2A T 541
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542 Figure 9.13 Electrophoresis in I percent agarose gels of nicked and closed circular PSMI DNA incubated with (a) cis- and (b) trans-[Pt(NH 3 hCl z ] as function of time. After electrophoresis, gels were stained with ethidium bromide. Reproduced with permission from Reference 51. produce changes in the superhelix density when bound to closed circular DNA. 87 As shown in Figure 9.13, increasing concentrations of platinum bound per nu- cleotide on the DNA first retard its mobility and then increase its mobility through the gel. These interesting alterations in gel mobility occur because the nega- tively coiled superhelix unwinds first into an open, or untwisted, form and then into a positively supercoiled form. The conformational changes, which are de- picted in Figure 9.14, are directly proportional to the drug-per-nucleotide, or (DlNh, ratio. In addition to superhelical winding, both platinum complexes in- crease the mobility of nicked circular DNA in the gels (Figure 9.13). Nicked DNA has one or more breaks in the sugar-phosphate backbone, which relieve the topological constraint and prohibit the DNA from twisting into superhelical structures. What could be the cause of these physical changes in the DNA structure upon cis- or trans-DDP binding? Intercalation can be excluded, not only be- cause the compounds do not have the aromatic character normally associated with intercalators (Figure 9.11), but also through studies of the manner by which these and other platinum complexes inhibit the intercalative binding of EtdBr to DNA. 89 ,90 Platinum metallointercalators such as [Pt(terpy)(HET)] + are compet- itive inhibitors of EtdBr binding, as measured by fluorescence Scatchard plots, whereas the non-intercalators cis- and trans-DDP are not. Moreover, intercala- tion tends to lengthen and stiffen the double helix, whereas the mobility changes of nicked circular DNAs upon binding of cis- or trans-DDP were shown by electron microscopy experiments to arise from a pronounced shortening of the DNA with increased Pt binding. One manner by which cis- or trans-DDP might produce these physical al- terations in DNA structure is by kinking the double helix at or near the binding site. Such an effect could be produced by the bidentate attachment of platinum; the monofunctional [Pt(dien)Cl] + complex does not have these pronounced ef-
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Negatively supercoiled DNA j 1 Relaxed DNA j 1 Positively supercoiled DNA Figure 9.14 Topological fonns of closed circular DNA. fects on DNA secondary structure. 91 Recently, it has been demonstrated that cis- DDP binding to DNA does indeed produce a pronounced bend in the helix axis.
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