Electrophoretic mobility of duplex DNA cross‐linked by mechlorethamine at a cytosine–cytosine mismatch pair

Abstract

The common nitrogen mustard, mechlorethamine, can form a covalent cross-link between the two bases of a cytosine-cytosine mismatch pair within a DNA duplex. The cross-linked species can be readily separated from DNA monoadducts and unreacted strands using denaturing polyacrylamide gel electrophoresis. Here, using DNA 19 mer duplexes that are mechlorethamine cross-linked at a C(4)-C(35), C(7)-C(32), C(10)-C(29), or C(13)-C(26) mismatch pair, we show that the denaturing polyacrylamide gel electrophoresis mobility of the cross-linked species is particularly sensitive to the proximity of the C-C cross-link to the duplex end. Species that are cross-linked at a C(4)-C(35) mismatch have greater mobilities than those cross-linked at C(7)-C(32) or C(13)-C(26), and the species with a central C(10)-C(29) cross-link have the lowest mobility. The mobility is also dependent on the proximity of the cross-link to a 5'-(32)P-phosphate or a 5'-fluorescein label. We interpret these results in terms of the conformational properties of the cross-linked species in the denaturing gel. The results are consistent with the retention of partial duplex character at the end proximal to the cross-link, with an influence on the mobility of the GC/AT ratio proximal to the cross-link and at the duplex end, and a small but discernible effect of the label.