RecA-catalyzed, sequence-specific alkylation of DNA by cross-linking oligonucleotides. Effects of length and nonhomologous base substitutions.
Oligodeoxyribonucleotides (ODNs) bearing the reactive nitrogen mustard chlorambucil have been used as sequence-directed affinity labeling reagents to investigate the length and homology requirements for RecA-catalyzed alkylation of double-stranded DNA. The cross-linkage reaction, which takes place at the N-7 position of a targeted complementary strand guanine following strand exchange, was highly sequence specific with both a 272 bp DNA fragment and a linearized plasmid. Alkylation required the ODN to be at least 26 nucleotides long and to possess homology 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 guanine. Longer heterology also inhibited alkylation when it prevented strand exchange. Our inability to detect cross-linkage in stable synaptic complexes 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 reaction specificity of chlorambucil-bearing ODNs suggests that they may have general use as recombinase-mediated DNA targeting agents.
Podyminogin
M A
MA
Epoch Pharmaceuticals, Inc., Bothell, Washington 98021, USA.
Meyer
R B
RB
Gamper
H B
HB
D000345:Affinity Labels / Q000378:metabolism; D000478:Alkylation; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D001483:Base Sequence; D002699:Chlorambucil / Q000378:metabolism / Q000494:pharmacology; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011938:Rec A Recombinases / Q000378:metabolism*; D016415:Sequence Alignment
D000345:Affinity Labels; D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D002699:Chlorambucil; D004247:DNA; D011938:Rec A Recombinases