DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence.
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.
Romero
R M
RM
Department of Pharmaceutical Sciences, University of Southern California, Los Angeles 90089-9121, USA.
Rojsitthisak
P
P
Haworth
I S
IS
D000477:Alkylating Agents / Q000737:chemistry / Q000378:metabolism; D001482:Base Composition; D020137:Base Pair Mismatch / Q000235:genetics*; D020029:Base Pairing; D001483:Base Sequence; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism*; D003596:Cytosine / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005600:Fragile X Syndrome / Q000235:genetics; D020862:GC Rich Sequence / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008466:Mechlorethamine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D013379:Substrate Specificity; D013696:Temperature
D000477:Alkylating Agents; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D015335:Molecular Probes; D008466:Mechlorethamine; D003596:Cytosine; D004247:DNA