MOLECULAR ANALYSIS OF SITE-SPECIFIC DNA RECOMBINATION

位点特异性DNA重组反应控制着许多类型的生物

Site-specific DNA recombination reactions control many types of biological reactions. These include many forms of gene regulation, viral integration and excision, chromosome partitioning and the combinatorial joining of gene segments to create new genes. While site-specific rearrangements can be part of the normal developmental program of specialized cells, aberrant rearrangements can lead to cancer and birth defects. The proposed work is concerned with understanding the molecular mechanisms involved in a model site-specific recombination reaction that regulates the alternative synthesis of flagellin proteins (flagellar phase variation) in Salmonella. This DNA inversion reaction can be studied in a purified in vitro system consisting of three proteins: Hin, Fis, and HU. The supercoiled DNA substrate must contain the two recombination sites plus a recombinational enhancer sequence, that together with the Fis protein, functions to increase the rate of inversion 102-103-fold in vivo and in vitro. Initiation of DNA strand exchange requires the formation of an invertasome structure in which the two recombination sites bound by the Hin recombinase are associated with the enhancer bound by Fis. Hin also has the ability to associate the two hix recombination sites into a complex, but without the enhancer, the paired-hix complex is incapable of supporting recombination. A variety of approaches will be used to discern the biochemical events that are required for invertasome assembly and strand exchange. We will attempt to determine if the paired-hix complex is a precursor to the invertasome and what molecular changes are triggered by the association of the enhancer. In order to do this, the protein interfaces that occur between and among Hin and Fis promoters in the different nucleoprotein complexes will be determined using mutational and site-directed crosslinking techniques. Some of these experiments will be guided by our initial mutation and crystal structure analysis of Fis as well as the published crystal structure of resolvase, which is related to Hin. Experiments are proposed which address the role of DNA supercoiling in invertasome assembly and recombination and to provide evidence that Hin-mediated recombination in vivo occurs in an invertasome structure. It has been proposed that DNA strand exchange is mediated by the rotation of Hin subunits in the invertasome complex. An attempt will be made to provide direct biochemical evidence for this mechanism. Both the Hin and Fis proteins specifically interact with DNA in novel ways. Fis binds to a highly degenerate DNA sequence and induces significant distortion of the DNA upon binding; stable Hin binding requires specific minor groove interactions. Intensive efforts to generate crystal structures of these proteins bound to DNA are ongoing. Mutagenesis and binding studies will also be used to specifically address Fis-DNA recognition and the nature and importance of Fis-induced DNA bending for enhancer function. The findings obtained from this work should be applicable to the many systems where the assembly of complex nucleoprotein complexes are required to promote various biological reactions.