MOLECULAR ANALYSIS OF SITE-SPECIFIC DNA RECOMBINATION

特定位点的DNA重排是复杂的反应

Site-specific DNA rearrangements are complex reactions in which nucleotide sequences located at great distances from one another are joined together in a highly precise manner. We are studying the flagellar phase variation system of Salmonella. Gene regulation is accomplished in this system by the site-specific inversion of a 1 kb segment of DNA. A cell-free system which supports the inversion reaction has been developed. Using this system, the protein and sequence determinants which are required for the reaction have been identified. Maximum rates of inversion require three proteins: the Hin recombinase, the histone-like protein HU, and a previously unidentified 12,000 D host protein (Factor II). The DNA substrate must be supercoiled and contain two recombination sites in the appropriate configuration, plus a 60 bp recombinational enhancer sequence. The recombinational enhancer, together with its associated protein (Factor II), can function at many different locations relative to the recombination sites to stimulate the inversion reaction over 150-fold. The proposed work will characterize the Hin inversion system, focusing in particular, on the mechanism of enhancer function in this system. Reaction intermediates and complexes formed during the reaction will be isolated and characterized, particularly with regard to the participation or role of the enhancer and Factor II protein. The gene for Factor II will be cloned for a detailed genetic and biochemical analysis. In addition, mutants of Hin which are defective in recombination will be isolated. The subsequent in vitro analysis of these mutant proteins will allow us to dissect the various steps in the reaction. Attempts will be made to isolate suppressors in the Factor II gene of specific mutations in hin to probe any interactions which may be occuring between Factor II and the Hin recombinase. Finally, the role of Factor II in the life of E. coli will be investigated and a search for other reactions involving Factor II acting via enhancer-like elements will be undertaken. These studies should both provide valuable information on the mechanism of site- specific DNA recombination and increase our understanding of complex protein-DNA reactions involving enhancers and DNA structure.