Front Cover; Fe-S Cluster Enzymes Part B; Copyright; Contents; Contributors; Preface; Chapter One: Iron-Sulfur Clusters in DNA Polymerases and Primases of Eukaryotes; 1. Introduction; 2. Iron-Sulfur Clusters in DNA Polymerases and Primase; 2.1. DNA Polymerases at the Replication Fork; 2.2. Iron-Sulfur Cluster in the Large Subunit of DNA Primase; 2.3. Iron-Sulfur Clusters in B-Family DNA Polymerases; 3. Genetic Evidence for the Important Roles of Iron-Sulfur Clusters in DNA Replication In Vivo; 4. Purification of Eukaryotic B-Family DNA Polymerases; 4.1. Procedure; 4.2. Notes.

5. Analysis of Iron Content in Protein Samples5.1. Procedure; 5.2. Notes; Acknowledgment; References; Chapter Two: Fe-S Clusters and MutY Base Excision Repair Glycosylases: Purification, Kinetics, and DNA Affinity Measurements; 1. Introduction; 2. Over Expression and Purification of MutY Homologs; 2.1. Considerations for Isolating MutY Homologs; 2.2. Bacteria as an Overexpression Host; 2.3. Purification of E. coli MutY; 2.4. Purification of a Thermophilic Homolog, G. stearothermophilus MutY; 2.5. MBP-MutY for Higher Yields and Solubility; 2.6. Bacterial Expression of M. musculus Mutyh.

2.7. Eukaryotic Expression System for Production of Homo sapiens MUTYH3. Gel-Based Adenine Glycosylase Assays and Measurements of Kinetic Parameters; 3.1. General Setup and Execution of the Glycosylase Assay; 3.1.1. Radiolabeling the DNA Substrate; 3.1.2. Preparation of a Denaturing Polyacrylamide Gel; 3.1.3. General Assay Setup; 3.1.4. Visualization and Quantitation of Results; 3.1.5. Salt Concentration in Assay Buffer; 3.2. Correcting the Enzyme Concentration for the Percent Active Fraction; 3.3. Determining the Rate of Product Release; 3.4. Assessing the Rate of Glycosidic Bond Cleavage.

4. Gel-Based Assays for Determining MutY-DNA Affinity4.1. General Features and Considerations of Gel-Based Binding Assays With MutY; 4.1.1. Radiolabeling the DNA Substrate; 4.1.2. Preparation of a Nondenaturing Polyacrylamide Gel; 4.1.3. Running an EMSA; 4.1.4. Visualization and Quantitation of Results; 4.2. Measurements of MutY-DNA Dissociation Constants; 4.3. Measurement of DNA Dissociation Rate (koff); 5. Application of Methods to Reveal Roles of the Fe-S Cluster Cofactor in MutY Homologs; Acknowledgments; References.

Chapter Three: Cellular Assays for Studying the Fe-S Cluster Containing Base Excision Repair Glycosylase MUTYH and Homologs1. Introduction; 2. Mutation Suppression Activity Measured in Rifampicin Resistance Assays; 2.1. Overview of the Rifampicin Resistance Assay; 2.2. Transformation and Growth of Cells; 2.3. Determining the Mutation Frequency; 2.3.1. Troubleshooting Tips; 2.4. Case Study: Use of Rifampicin Resistance Assay to Characterize Zn-Linchpin Motif in MUTYH; 3. Analysis of MutY-Mediated Repair of Defined Plasmid Substrates in E. coli.