RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange

Abstract

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.