Nucleic Acids Research, 2001, Vol. 29, No. 22 4716-4723
© 2001 Oxford University Press
Extrahelical cytosine bases in DNA duplexes containing d[GCC]n·d[GCC]n repeats: detection by a mechlorethamine crosslinking reaction
Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA
The cytosine–cytosine (C–C) pair is one of the least stable DNA mismatch pairs. The bases of the C–C mismatch are only weakly hydrogen bonded, and previous work has shown that, in certain sequence contexts, they can become unstacked from the core helix, and adopt an ‘extrahelical’ location. Here, using DNA duplexes with d[GCC]n·d[GCC]n fragments containing C–C mismatches in a 1,4 bp relationship, we show that cytosine bases of different formal mismatch pairs can be crosslinked by mechlorethamine. For example, in the duplex d[CTCTCGCCGCCGCCGTATC]·d[GATACGCCGCCGCCGAGAG], where underlined cytosine bases are present as the formal C–C mismatch pairs C7–C32, C10–C29 and C13–C26, we show that two mechlorethamine crosslinks form between C13 and C29 and between C10 and C32, in addition to crosslinks at C7–C32, C10–C29 and C13–C26 (we have reported previously the crosslinking of formal C–C pairs by mechlorethamine). We interpret the formation of the C13–C29 and C10–C32 crosslinks as evidence of an extrahelical location of the crosslinkable cytosines. Such extrahelical cytosine bases have been observed previously for a single C–C mismatch pair (in the so-called E-motif conformation). In the E-motif, the extrahelical cytosines are folded back towards the 5'-end of the duplex, consistent with our crosslinking data, and also consistent with the absence of C7–C29 and C10–C26 crosslinks in the current work. Hence, our data provide evidence for an extended E-motif DNA (eE-DNA) conformation in short d[GCC]n·d[GCC]n repeat fragments, and raise the possibility that such structures might occur in much longer d[GCC]n·d[GCC]n repeat tracts.
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