Ma. Podyminogin et al., RECA-CATALYZED, SEQUENCE-SPECIFIC ALKYLATION OF DNA BY CROSS-LINKING OLIGONUCLEOTIDES - EFFECTS OF LENGTH AND NONHOMOLOGOUS BASE SUBSTITUTIONS, Biochemistry, 35(22), 1996, pp. 7267-7274
Oligodeoxyribonucleotides (ODNs) bearing the reactive nitrogen mustard
chlorambucil have been used as sequence-directed affinity labeling re
agents to investigate the length and homology requirements for RecA-ca
talyzed alkylation of double-stranded DNA. The cross-linkage reaction,
which takes place at the N-7 position of a targeted complementary str
and guanine following strand exchange, was highly sequence specific wi
th both a 272 bp DNA fragment and a linearized plasmid. Alkylation req
uired the ODN to be at least 26 nucleotides long and to possess homolo
gy to the target in the vicinity of the modification site, The extent
of alkylation was improved by using longer ODNs, with a 50-mer giving
over 50% reaction. Mismatches inhibited alkylation when they perturbed
the structure of the strand exchange product near the targeted guanin
e. Longer heterology also inhibited alkylation when it prevented stran
d exchange. Our inability to detect cross-linkage in stable synaptic c
omplexes unable to undergo complete strand exchange is best explained
by a model for homologous alignment in which the presynaptic filament
approaches from the minor groove of the duplex. Since the N-7 position
of guanine is in the major groove, it is inaccessible to the tethered
chlorambucil group of the ODN during the search for homology. The rea
ction specificity of chlorambucil-bearing ODNs suggests that they may
have general use as recombinase-mediated DNA targeting agents.