title: Extrahelical cytosine bases in DNA duplexes containing d[GCC](n).d[GCC](n) repeats: detection by a mechlorethamine crosslinking reaction.
abstract: 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 C(7)-C(32), C(10)-C(29) and C(13)-C(26), we show that two mechlorethamine crosslinks form between C(13) and C(29) and between C(10) and C(32), in addition to crosslinks at C(7)-C(32), C(10)-C(29) and C(13)-C(26) (we have reported previously the crosslinking of formal C-C pairs by mechlorethamine). We interpret the formation of the C(13)-C(29) and C(10)-C(32) 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 C(7)-C(29) and C(10)-C(26) 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90089-9121, USA.
authors.forename: P
authors.initials: P
authors.lastname: Rojsitthisak
authors.forename: R M
authors.initials: RM
authors.lastname: Romero
authors.forename: I S
authors.initials: IS
authors.lastname: Haworth
mesh_terms: D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D003432:Cross-Linking Reagents; D003596:Cytosine / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008466:Mechlorethamine / Q000737:chemistry; D009690:Nucleic Acid Conformation; D018911:Trinucleotide Repeats
chemical_list: D003432:Cross-Linking Reagents; D008466:Mechlorethamine; D003596:Cytosine; D004247:DNA