Sequence-specific covalent modification of DNA by cross-linking oligonucleotides. Catalysis by RecA and implication for the mechanism of synaptic joint formation

Abstract

Oligodeoxynucleotides (ODNs) were conjugated to chlorambucil and used as affinity labeling reagents to study joint molecule formation by the Escherichia coli recombinase recA. Chlorambucil is a bifunctional nitrogen mustard which alkylates the N-7 position of guanine in the major groove of double-stranded DNA (dsDNA). Incoming ODNs at least 30 nucleotides long cross-linked to a long homologous duplex DNA in the presence of recA and ATP gamma S. Efficient cross-linkage to the complementary recipient strand of the joint occurred preferentially at guanines positioned 5' relative to the appended chlorambucil group. The pattern of recipient strand alkylation was identical to that observed within a protein-free duplex and indicated that strand exchange had occurred prior to alkylation. Modification of the outgoing homologous strand of the joint was less efficient and spanned a 15-20 nucleotide long region offset to the 3' side of the tethered chlorambucil. Alkylation of both recipient and outgoing strands in the same joint molecule occurred with low frequency. By contrast, no affinity alkylation of the displaced strand was observed within a synthetic D-loop. These reaction patterns suggest that the incoming ODN approaches from the minor groove of the duplex to yield a poststrand exchange joint in which the major groove of the newly formed heteroduplex harbors the outgoing strand in an unpaired state. No evidence was obtained for the involvement of a triple-stranded DNA intermediate in recombination.