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Fig. 4.6.6. Assay for the spectroscopic investigation of the distance-dependence of HT in DNA using the protein M.Hhal (Trp 237 mutant).

tioned above [19]. The rate of formation of the radical intermediates was measured as a function of the distance using three different DNA substrates (M1, M2, and M3; Figure 4.6.6). The CT reaction was observed by time-resolved transient absorption spectroscopy. The product radical was identified to be a mixture of the Trp and G radicals occurring in the DNA-protein contact area. The laser experiments revealed that the signals showed little variation in the rate of formation of the transient radical in the three different DNA substrates. Biexponential fitting yielded rate constants of ~5 x 106 s-1 and ~3 x 105 s-1 for all duplexes. This clearly establishes a lower limit for HT in DNA of >106 s-1 through 50 A of the base stack. On the basis of the absence of significant distance dependence we concluded that HT through the DNA is not a rate-limiting step.

Given the results from protein-modulation described in this section, DNAmediated charge-transfer chemistry requires consideration biologically and physiologically. It has, for example, been suggested that regions containing a large amount of G are typically found in CpG islands, introns, and telomeres and, therefore, that such areas are hot spots for G damage, and as a result, could prevent the genome from oxidative radical damage [21].

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