Structural and energetic aspects of protein DNA-recognition

The project aims at using cod Uracil-DNA glycosylase (cUNG) as a model system in the study of DNA recognition and cold activity. The cod UNG possesses typical cold adaptation features such as increased catalytic efficiency and reduced temperatur stability compared to human UNG. The crystal structure of cod UNG has recently been determined and shows similarity to the structures of human, herpes simplex virus and E.coli UNG. Furthermore, in the order of 30 mutants has so far been constructed and characteriz ed at various levels. Characterization of mutants of cUNG in combination with structure analysis indicates a highly fine tuned mechanism responsible for the increased catalytic efficiency. Analysis of mutants with substitutions in loop regions and altera tions of the surface charge indicate that an increased local flexibility and an optimisation of the electrostatic potential cause the increased activity of the enzyme. These observations wil l be further explored by the use of crystal structure analysis c ombined with theoretical calculations on cUNG-DNA/inhibitor complexes. Crystal structure analysis and calculations will be applied on both complexes with native enzyme and on a series of muta nts. The results from the study are expected to provide increas es insight into the molecular basis of protein-DNA interaction and cold activity.