The long-term goal of this project is to understand both how proteins recognize specific DNA sequences, and how the recognition mechanisms are varied to accommodate the functional requirements of the protein. To that end, a combination of mutant characterization and protein biochemistry will be used to study three functionally disparate DNA-binding proteins which all come from the Pvu II restriction-modification system. The first of these proteins is the Pvu II DNA methyltransferase, which binds S-adenosylmethionine and the DNA duplex sequence CAGCTG and methylates the amino group of the internal cytosine. The second protein is the Pvu II restriction endonuclease, which binds the same DNA sequence and Mg++ and catalyzes phosphodiester cleavage between the central two bases. The third protein is the Pvu II C protein, which appears to have a more degenerate recognition specificity on the DNA and activates transcription of the endonuclease gene. The immediate goals are to identify the domains and amino acids that play key roles in recognizing the substrate DNA sequence and the other substrates as well. Understanding sequence-specific recognition of DNA by proteins, particularly when a catalytic center must be brought into proximity with the DNA, is an important goal both for a sophisticated understanding of gene expression and for the further development of biotechnology. %%% The two long-term goals of this project are to understand how proteins recognize specific DNA base sequences despite the large excess of nonsubstrate DNA in the cell, and how these sequence recognition mechanisms vary to accommodate the other functional requirements of the protein when a catalytic center must be brought close to the DNA to carry out reactions. Understanding sequence- specific recognition of DNA by proteins is an important goal both for fully understanding the control of gene expression, and for the further development of biotechnology. In this study, a combination of mutagenesis and protein biochemistry will be used to study three DNA-binding proteins which all come from the Pvu II restriction- modification system but which each have very different fuctions. The first of these proteins is the Pvu II DNA methlytransferase, which binds S-adenosylmethionine and the DNA duplex sequence CAGCTG and methylates the amino group of the internal cytosine. The second protein is the Pvu II restriction endonuclease, which binds the same DNA sequence an Mg++ and catalyzes phosphodiester cleavage between the central two bases. The third protein is the Pvu II C protein, which appears to bind the Pvu II DNA and activate transcription of the endonuclease gene. The immediate goals are to identify the domains and amino acids of these three proteins that play key roles in recognizing the substrate DNA sequence, in recognizing the other substrates, and in carrying out the chemical reactions of methylation or cleavage.
