| R.M.Romero, M.Mitas and I.S.Haworth. Anomalous Crosslinking by Mechlorethamine of DNA Duplexes Containing C-C Mismatch Pairs. Biochemistry, 1999, 38, 3641-3648. |
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Nitrogen mustards
such as mechlorethamine have previously been shown to covalently crosslink
DNA through the N7 position of the two guanine bases of a d[GXC]·d[GYC]
duplex sequence, a so-called 1,3 G-G-crosslink, when X-Y = C-G or T-A.
Here, we report the formation of a new mechlorethamine crosslink with the
d[GXC]·d[GYC] fragment when X-Y is a C-C mismatch pair. Mechlorethamine
crosslinks this fragment preferentially between the two mismatched cytosine
bases, rather than between the guanine bases. The crosslink also forms
when one or both of the guanine bases of the d[GCC]·d[GCC] fragment
are replaced by N7-deazaguanine, and, more generally, forms with any C-C
mismatch, regardless of the flanking base pairs. Piperidine cleavage of
the crosslink species containing the d[GCC]·d[GCC] sequence gives
DNA fragments consistent with alkylation at the mismatched cytosine bases.
We also provide evidence that the crosslink reaction occurs between the
N3 atoms of the two cytosine bases by showing that the formation of the
C-C crosslink is pH dependent for both mechlorethamine and chlorambucil.
Dimethyl sulfate (DMS) probing of the crosslinked d[GCC]·d[GCC]
fragment showed that the major groove of the guanine adjacent to the C-C
mismatch is still accessible to DMS. In contrast, the known minor groove
binder Hoechst 33258 inhibits the crosslink formation with a C-C mismatch
pair flanked by A-T base pairs. These results suggest that the C-C mismatch
is crosslinked by mechlorethamine in the minor groove. Since C-C pairs
may be involved in unusual secondary structures formed by the trinucleotide
repeat sequence d[CCG] n, and associated with triplet repeat
expansion diseases, mechlorethamine may serve as a useful probe for these
structures.
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