A genetic approach will be used to determine the chemical contacts made between Saccharomyces cerevisiae DNA-binding proteins and their specific recognition sites. We will select subclones of yeast genomic DNA and cDNA's in phage or plasmid vectorsin S. typhimurium and E. coli hosts) that produce functional repressors of cloned substitutions for the Salmonella phage P22 Mnt operator on """"""""challenge phages."""""""" These substitutions include sites involved in the regulation of mating type, chromosome and plasmid replication and stability, the expression of genes under general amino acid control, and the expression of genes in response to heat shock control, as well as sites that occur frequently as short, repeated polymers in the cerevisiae genome. In collaboration with other research groups, genes encoding site-specific binding activities will be subdefined on clones, sequenced, and expressed in heterologous prokaryotic systems. Altered specificity mutations will be isolated to define amino acid residues of the DNA-binding proteins directly involved in sequence-specific recognition. Once we have demonstrated specific binding in vivo, we will attempt to develop in vitro binding assays to purify these proteins. We will attempt to transduce, or """"""""convert,"""""""" site and protein mutations onto the haploid and diploid cerevisiae genomes to try to determine the importance of specificity to the roles of these genetic elements in vivo. The long-term goal of this research is a chemical understanding of how proteins recognize specific sequences on DNA molecules. Analysis of these interactions should begin to reveal the nature of the recognition code (the set of specific weak chemical bonds) that underlies DNA/protein interactions and to test whether this code is universal, i.e., the same in eukaryotes as in prokaryotes. Moreover, we will demonstrate that the challenge phage selection for DNA-binding proteins may be extended to any specific site; that is, any site can be used to """"""""fish out"""""""" the proteins involved in its recognition. Future applications of this selection to higher eukaryotes will circumvent current, more work-intensive approaches to define specific regulatory interactions.
| Lebreton, B; Prasad, P V; Jayaram, M et al. (1988) Mutations that improve the binding of yeast FLP recombinase to its substrate. Genetics 118:393-400 |
| Hughes, K T; Youderian, P; Simon, M I (1988) Phase variation in Salmonella: analysis of Hin recombinase and hix recombination site interaction in vivo. Genes Dev 2:937-48 |
