When a DNA region is protected by the presence of a molecule, chemical digestion is blocked and a ��gap�� is present on the autoradiograph. In the control (CTR, Figure 2), all fragments are present with broadly the same signal intensity Ganetespib purchase on the gel, while in all the treated sample lanes a typical ��gap�� is common in AT-rich regions. The brackets highlight these regions. The distamycin A backbone present in the brostallicin chemical structure drives the DNA interaction towards AT-rich regions in the same way as previously shown for tallimustine. In fact, brostallicin shows a noncovalent DNA interaction effect superimposable to that of tallimustine and distamycin A (internal positive control). These regions are highlighted by brackets. The differences in band intensities were due to differences in gel loading.
Figure 2 MPE footprinting analysis of the SV40 DNA plasmid (751-bp, panel A; and 4492-bp, panel B) treated with distamycin A (DISTA), tallimustine (TAM), and brostallicin as described in Materials and Methods section. CTR=untreated control DNA. Brackets … On the basis of the previously reported data showing that brostallicin is able to covalently interact with DNA upon in vitro reduction by the GSH/GST system (Geroni et al, 2002), we further tested this hypothesis by incubating the drug-DNA solution with and without GSH and GST in an in vitro system. The sequence-specific, covalent DNA interaction of brostallicin in comparison with tallimustine was analysed by the Taq polymerase stop assay.
This assay is a linear amplification method employing the properties of DNA polymerase to investigate the sequence selectivity of the interaction between DNA-damaging agents and the DNA. As expected, tallimustine retained its high sequence specificity in alkylating DNA at the target hexamer (5��-TTTTGA), while brostallicin per se was completely unable to produce any alkylation in the selected DNA region (Figure 3). Brostallicin alone did not alkylate DNA, while a band was observed when GST/GSH was added to the reaction mixture. In the absence of brostallicin, GSH/GST did not induce any alteration able to block Taq polymerase. It is important to underline the fact that, although the interaction of brostallicin with DNA involves AT-rich regions, the compound binds to a sequence (AAAG) different from those previously reported for tallimustine.
Studies are still in progress to better define the sequence of the alkylated regions. Figure 3 Autoradiography Anacetrapib of a typical Taq Stop Assay on topoisomerase II cDNA after treatment with brostallicin and tallimustine (TAM). The experiment was performed as described in Materials and Methods section. CTR=control untreated sample. Arrows indicate … Loss of MLH1 or MSH2 does not alter sensitivity to brostallicin The question was addressed as to whether loss of either MLH1 or MSH2 affects the sensitivity to brostallicin using the clonogenic assay.