[Frontiers in Bioscience 18, 224-240, January 1, 2013]

Single-molecule and bulk approaches to the DnaB replication fork helicase

Daniel L. Kaplan1, Omar A. Saleh2, Noah Ribeck3

1Department of Biological Sciences, Vanderbilt University, VU Station B, Box 35-1634, Nashville TN 37235, 2Materials Dept., University of California, Santa Barbara CA 93106, 3Department of Microbiology and Molecular Genetics, Michigan State University, 2215 Biomedical Physical Sciences, East Lansing MI 48824

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Review of single-molecule manipulation approaches to motor proteins
3.1. SMM instrumentation
3.2. Tether design for SMM
4. Comparing and contrasting bulk and SMM measurements of the helicase DnaB
4.1. Bulk characterization of DnaB
4.1.1. Purification and Initial Characterization of E. coli DnaB
4.1.2. It is established that DnaB is a helicase using purified protein and radiolabeled DNA
4.1.3. Domain function of DnaB is elucidated
4.1.4.Fluorescent studies demonstrate that DnaB binds to 20 nucleotides of ssDNA
4.1.5. Rapid quench flow technique reveals mechanism of ATP hydrolysis
4.1.6. Electron microscopy shows that single-stranded DNA passes through the central channel of T7 gp4
4.1.7. Fluorescent energy transfer show that single-stranded DNA passes through the central channel of DnaB
4.1.8. Fluorescence studies reveal kinetics of DnaB-catalyzed unwinding
4.1.9.Electron microscopy studies of DnaB reveal its shape
4.1.10. Crystal structures of T7 gp4 and DnaB reveal information about helicase mechanism
4.1.11. My (Kaplan) studies of DnaB
4.1.12. DnaB functions with other proteins
4.2. Single-molecule characterization of DnaB
5. Summary and perspective
6. Acknowledgments
7. References

1. ABSTRACT

Motor proteins are enzymes that accomplish mechanical work in a wide variety of biological processes. In this review we focus on bulk and single molecule methods to study how motor proteins function. We discuss in detail the analysis of the motor protein DnaB, a hexameric helicase that unwinds DNA at a replication fork in Gram-negative bacteria. Bulk and single-molecule studies have complemented one another to arrive at a comprehensive mechanistic view of how DnaB unwinds double-stranded DNA.