CAREER: Structure and Function in Restriction Endonucleases

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Dupureur98759171. TECHNICALRestriction enzymes are the simplest biological agents ofsite-specific nucleic acid chemistry and as such serve as molecularlaboratories for dissecting natural activities and engineering new ones.In spite of the importance of the metal ion dependence of restrictionenzyme mechanisms, it is unclear how many metal ions bind these enzymes. In order to propose relevant reaction mechanisms, detailed measurements of metal ion affinity, stoichiometry, and substrate coordination in restriction enzymes must first be developed and conducted in a direct and comprehensive fashion. As evidenced by the null activity of enzymes mutated distal to the active site, metal ion-mediated catalysis alsoappears to be tightly coupled to conformational changes, which prompted the hypothesis that these mutations compromise critical metal ion-substrate interactions in the active site. This study applies an unique combination of instrumental methods and international, industrial, and academic collaborations to the issues of metal ion binding and conformational behavior in restriction endonucleases. To better understand underlying themes in endonuclease function, two representative enzymes will be compared. The first specific aim of this study is to determine the affinities and binding stoichiometries for required divalent metal ions. These experiments are coupled to studies of the importance of metal ion-substrate interactions in the active site, using a combination of isothermal titration calorimetry (ITC), site-directed mutagenesis, phosphorothioate substitution, and paramagnetic resonance methods. The staggered-end cutter EcoRI endonuclease and the blunt-end cutter PvuII endonuclease serve as the representative restriction endonucleases. The second specific aim is to examine the extent and nature of metal ion and substrate-induced conformational changes in restriction enzymes. Isotopic labeling and multidimensional nuclear magnetic resonance (NMR) methods are applied to PvuII endonuclease, the smallest type II restriction enzyme yet characterized. Parallel to the execution of research plans, two advanced graduate courses will be developed. The first course introduces advanced spectroscopic methods to the study of protein structure and function and serves a growing and vital biophysical chemistry community on campus. Complementary to this is a course which develops the scientific writing skills among advanced biochemistry graduate students through a discussion of both the structure and language of proposals and involves the participation of both academic and industrial collaborators.2. Non-technical Restriction enzymes are proteins that recognize and cut specificsequences of DNA with the assistance of metal ions. These "molecularscissors" are powerful tools of biotechnology. There is considerableinterest in expanding the repertoire of these enzymes, that is, developing new enzymes with new specificities. The approach of this study is to understand how existing enzymes work. This study is divided into parts: First, to understand how the metals ion help the enzyme cut DNA, comparing the metal ion binding properties of two representative restriction enzymes, PvuII endonuclease and EcoRI endonuclease. Instrumental methods are applied to determine how many metal ions bind each enzyme, where they bind, and how the enzyme-bound metal ions interact with the DNA. This helps us understand what is required for DNA cutting. The question of how the conformation or shape of PvuII endonuclease changes when it interacts with metal ions and different DNA sequences is also studied. To accomplish this, nuclear magnetic resonance (NMR) spectroscopy, an instrumental method which is sensitive to slight changes in enzyme conformation is applied. Together, these two aims help us understand how DNA sequence recognition and metal ion-assisted DNA cleavage are related in these enzymes. Concurrent with and complementary to these research activities, two new elective graduate courses will be developed. The first is a survey of spectroscopic methods as applied to problems in protein structure-function and will involve faculty members with expertise in this area. The other elective is a course in the structure and language of research proposals and will include discussions led by study sectionmembers and scientists from industry.