DNA Interstrand Crosslink Formation by Mechlorethamine at a Cytosine–Cytosine Mismatch Pair: Kinetics and Sequence Dependence

Abstract

Expansion of the triplet repeat DNA sequence d[CGG]n · d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G–G and C–C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C–C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C–C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C–C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4] · d[N4N3N2N1Cm1m2m3m4] sequence, where M–m and N–n are complementary base pairs, the amount of crosslink increased with increasing G–C content of the eight base pairs neighboring the C–C mismatch and with the proximity of the G–C pairs to the C–C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C–C pair flanked by G–C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C–C pair flanked by A–T base pairs, the simulations suggested local opening of the duplex around the C–C pair, making it a less effective target for mechlorethamine.