An Assay for In Vitro Recombination Between Duplex DNA Molecules

The RecA protein of Escherichia coli is essential for genetic recombination and has been extensively characterized (1 –3 ). In vitro, purified RecA protein is able to promote recombination reactions of two types: (i) strand transfer between circular single-stranded DNA (ssDNA) and homologous linear duplex DNA and (ii) strand exchange between circular duplex DNA with a defined single-stranded gap and homologous linear duplex DNA (Fig. 1A ) In this chapter, we describe the preparation of substrates for reaction (ii), which occurs between essentially duplex DNA molecules. The reaction has been used extensively in studies of the mechanism of RecA-mediated strand exchange (4 ,5 ) and may also be used to assay for activities capable of resolving Holliday junctions in DNA (6 ,7 ).