E COLI SINGLE-STRANDED DNA BUILDING PROTEIN

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Single-stranded DNA binding proteins are of central importance in DNA replication, recombination and repair. In general, these proteins bind tightly to single-stranded DNA and have no known catalytic properties of their own. SSB from E. coli is one such protein that is of particular interest and importance due in large measure to the advanced state of knowledge of basic DNA enzymology and the ability to perform detailed genetic analyses in that system. In this regard the E. coli system uniquely qualifies as a model for the mechanistic analysis of corresponding proteins from higher organisms. SSB is a basically simple protein and a great deal is known about its physicochemical properties and the genetic effects which result from its deficiency as well as certain in vitro biochemical reactions, particularly DNA replication, in which it participates. In spite of this knowledge, however, we do not understand in any detailed biochemical sense the mechanism of action of this protein. Its deceptive simplicity contrasts its intimate involvement in every aspect of DNA metabolism. It is, therefore, of basic importance that we understand how this and similar proteins actually function and to this end we will perform the following studies. The crystal structure of SSB and certain variant SSB's will be determined. Mutations which suppress the phenotype of ssb mutant strains will be studied in order to determine if alterations in other proteins might compensate for a deficiency in SSB. We will study in vivo and in vitro properties of several SSB-like proteins from transmissible plasmids which have recently been identified. Monoclonal antibodies to SSB will be studied. Both SSB and RecA affinity columns will be used to attempt to identify proteins which may interact with SSB and to specifically investigate the interaction of SSB and recA protein. Further attempts to elucidate interactions of SSB, RecA and single-stranded DNA will be made by studying the effect SSB has on certain reactions of RecA and ssDNA (e.g., D-loop formation). Finally, it is our aim to utilize the combined information from all of these studies to logically design variants of wild type E. coli SSB which can be used to test various ideas about the functional domains of SSB and their involvement in the biochemical mechanism of action of the protein.