In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step: strand exchange between homologous parental DNAs. In vitro, the product of this strand exchange reaction is a joint molecule composed of a single-strand DNA joined to one end of a linear duplex DNA. We have established a new paradigm for this homologous pairing, in essence, that recombinases such as the E. coli recA protein can hybridize a single strand of any sequence to an intact duplex. That is, the three strands form a novel DNA triplex (R-form DNA) in which the third strand may include both purines and pyrimidines. We have carried out three critical tests of the structural model of R-form DNA. First, using chemical footprinting we have demonstrated for the first time the hydrogen bonding between the third strand and the identical strand in the Watson-Crick duplex in homologous recombination. Second, using duplexes containing chemically substituted bases we have proven the importance of certain atoms to the thermal stability of R-form DNA. Finally, we have developed a method to selectively cleave duplex genomic DNA based on our ability to form R-form DNA: RecA Assisted Restriction Endonuclease (RARE) cleavage.
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