Homologous recombination plays a key role in generating genetic diversity,while maintaining protein functionality. The mechanisms by which RecA enables asingle-stranded segment of DNA to recognize a homologous tract within a wholegenome are poorly understood. The scale by which homology recognition takesplace is of a few tens of base pairs, after which the quest for homology isover. To study the mechanism of homology recognition, RecA-promoted homologousrecombination between short DNA oligomers with different degrees of heterologywas studied in vitro, using fluorescence resonant energy transfer. RecA candetect single mismatches at the initial stages of recombination, and theefficiency of recombination is strongly dependent on the location anddistribution of mismatches. Mismatches near the 5' end of the incoming strandhave a minute effect, whereas mismatches near the 3' end hinder strand exchangedramatically. There is a characteristic DNA length above which the sensitivityto heterology decreases sharply. Experiments with competitor sequences withvarying degrees of homology yield information about the process of homologysearch and synapse lifetime. The exquisite sensitivity to mismatches and thedirectionality in the exchange process support a mechanism for homologyrecognition that can be modeled as a kinetic proofreading cascade.
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