We found that several major chromosomal fragile sites in human
lymphomas, including the bcl-2 major breakpoint region, bcl-1 major translocation cluster, and c-Myc exon 1-intron 1 boundary, contain distinctive sequences of consecutive cytosines exhibiting a high degree of reactivity with the structure-specific chemical probe
bisulfite. To assess the inherent structural variability of duplex
DNA in these regions and to determine the range of structures reactive to
bisulfite, we have performed
bisulfite probing on genomic
DNA in vitro and in situ; on duplex
DNA in supercoiled and linearized plasmids; and on
oligonucleotide DNA/
DNA and
DNA/2'-O-methyl
RNA duplexes.
Bisulfite is significantly more reactive at the frayed ends of
DNA duplexes, which is expected given that
bisulfite is an established probe of
single-stranded DNA. We observed that
bisulfite also distinguishes between more subtle sequence/structural differences in duplex
DNA. Supercoiled plasmids are more reactive than linear
DNA; and sequences containing consecutive cytosines, namely GGGCCC, are more reactive than those with alternating
guanine and
cytosine, namely GCGCGC. Circular dichroism and x-ray crystallography show that the GGGCCC sequence forms an intermediate B/A structure. Molecular dynamics simulations also predict an intermediate B/A structure for this sequence, and probe calculations suggest greater
bisulfite accessibility of
cytosine bases in the intermediate B/A structure over canonical B- or
A-form DNA. Electrostatic calculations reveal that consecutive
cytosine bases create electropositive patches in the major groove, predicting enhanced localization of the
bisulfite anion at homo-C tracts over alternating G/C sequences. These characteristics of homo-C tracts in duplex
DNA may be associated with
DNA-
protein interactions in vivo that predispose certain genomic regions to
chromosomal fragility.