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 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 and an intact duplex. That is, the three strands form a novel DNA triplex (R-form DNA) in which the third strand may have any arbitrary sequence and must have a parallel orientation with respect to the phosphodiester backbone of the identical strand in the duplex. We have also been able to isolate synaptic complexes consisting of all the three strands and RecA. These structures have been studied in detail and will continue to be the basis of additional structural investigations. The kinetics of formation of these complexes have been used as a model for the homology search process in the obligatory recombination events during meiosis. The data shows that the search for homology is a very fast step that is not rate-limiting and that this is followed by a very slow step involving conformational changes of the protein and the DNA. Several of the rate constants in this reaction have been measured. Evaluating these rate constants has allowed us to delimit the possible structures involved at each step. In order to understand the mechanism and structures involved in greater detail we have determined that a 20 amino acid peptide from RecA has some of the activities of RecA. Efforts are now underway to determine the three-dimensional structure of this peptide when bound to DNA. The synaptic complexes have also been used to develop a method for the selective cleavage of human DNA (RecA-Assisted Restriction Endonuclease (RARE) cleavage). This method has been shown to be useful to map across """"""""gaps"""""""" in physical maps and t excise the very ends of chromosomes, that is telomeres and subtelomeric DNA. Most recently, we are using this technique to map across several gaps in the completed physical map of chromosome 21 and to map and excise centromeres from human chromosomes.
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