Enzymatic activities which act upon DNA have been studied extensively as to their in vitro characteristics, but it is only recently that their in vivo function and catalytic activities have been investigated. Our hope is to continue to study DNA enzymes, notably those from E. coli, where genetic tools aid us. The systems are: (1) The E. coli B modification and restriction enzymes. This pair of enzymes, a DNA methylase and DNA endonuclease-ATPase, respectively, are unique in that they are quite specific, share a common subunit, and have a complex mechanism. We are particularly interested in how the enzymes recognize DNA specifically, and exactly what is the function of the DNA dependent ATPase of the restriction enzyme. We also hope to gain insight into how the enzyme breaks DNA at sites other than the recognition site, by looking at how reaction perturbations affect this "binding." (2) The recBC DNase. This enzyme, from E. coli is involved in genetic recombination and in maintaining cell viability. It also has a DNA-dependent ATPase, and we hope to show that this activity is responsible for "melting" the DNA. We hope to demonstrate recombination directly in vitro with this melting reaction. (3) DNA Repair endonucleases. We have discovered several nucleases that are specific for damaged DNA. One recognizes minor photoproducts, the other apurinic sites, (but it is not endonuclease II). We expect to determine the exact specificity of these enzymes, and learn how they are utilized for repair.
NIH ExPorter
NIH ExPorter