In JoVE (1)
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Articles by F. Noah Biro in JoVE
Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein F. Noah Biro1, Jie Zhai1, Christopher W. Doucette1, Manju M. Hingorani1 1Molecular Biology and Biochemistry Department, Wesleyan University Msh2-Msh6 is responsible for initiating repair of replication errors in DNA. Here we present a transient kinetics approach towards understanding how this critical protein works. The report illustrates stopped-flow experiments for measuring the coupled DNA binding and ATPase kinetics underlying Msh2-Msh6 mechanism of action in DNA repair.
Other articles by F. Noah Biro on PubMed
Mechanism of Cadmium-mediated Inhibition of Msh2-Msh6 Function in DNA Mismatch Repair Biochemistry. Oct, 2009 | Pubmed ID: 19320425 The observation that Cadmium (Cd(2+)) inhibits Msh2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd(2+) binding to specific sites on Msh2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd(2+) binding to Msh2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity. Global fitting of the data to a multiligand binding model revealed that binding of about 100 Cd(2+) ions per Msh2-Msh6 results in its inactivation. This finding indicates that the inhibitory effect of Cd(2+) occurs via a nonspecific mechanism. Cd(2+) and Msh2-Msh6 interactions involve cysteine sulfhydryl groups, and the high Cd(2+):Msh2-Msh6 ratio implicates other ligands such as histidine, aspartate, glutamate, and the peptide backbone as well. Our study also shows that cadmium inactivates several unrelated enzymes similarly, consistent with a nonspecific mechanism of inhibition. Targeting of a variety of proteins, including Msh2-Msh6, in this generic manner would explain the marked broad-spectrum impact of Cd(2+) on biological processes. We propose that the presence of multiple nonspecific Cd(2+) binding sites on proteins and their propensity to change conformation on interaction with Cd(2+) are critical determinants of the susceptibility of corresponding biological systems to cadmium toxicity.