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

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 C₄–C₃₅, C₇–C₃₂, C₁₀–C₂₉, or C₁₃–C₂₆ 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 C₄–C₃₅ mismatch have greater mobilities than those cross‐linked at C₇–C₃₂ or C₁₃–C₂₆, and the species with a central C₁₀–C₂₉ cross‐link have the lowest mobility. The mobility is also dependent on the proximity of the cross‐link to a 5′‐³²P‐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.