CMU-Q-Department of Biological Sciences - Carnegie Mellon University

CMU-Q Students 2012

Hadya ElShakh, Hiba Al-Ashtal, Mei ElGindi, Raji Katibe, and Ridin Balakrishnan

Mutations in the Metal Ion Binding Site of EcoRV

This project serves to identify the effect of metal ion binding, by relieving strain between the DNA and the EcoRV endonuclease, on the dynamics of the protein while bound to DNA. This will provide insight into the dynamics of similar DNA binding proteins such as other endonucleases, polymerases and repressors. The goal is to use Nuclear Magnetic Resonance (NMR) Spectroscopy to visualize the dynamics of EcoRV while it is bound to DNA. While X-Ray Crystallography provides a visual representation of the enzyme bound to DNA, its shortcoming is that it does not allow for the dynamic changes in EcoRVs conformation to be studied because the protein must be in a crystal form. Single amino acid mutations in the EcoRV divalent cation binding sites were generated using PCR Mutagenesis. This serves two purposes, hindering the rapid cleavage of DNA once EcoRV is bound and reduces electrostatic strain between the negatively charged side chain and the negative DNA. By making these mutations we can visualize the effect that the reduction in strain caused by each mutation has on the binding dynamics of EcoRV. DNA encoding mutant EcoRV were incorporated into the pET expression vector and the gene expression protein products were isolated for analysis. The study of the mutated EcoRV in its bound state to DNA will provide us with information about metal ion binding by similar proteins that interact with DNA.