Studies of DNA-Replication at the Single Molecule Level Using Magnetic Tweezers

The development of tools to manipulate single biomolecules has opened a new vista on the study of many cellular processes. In this review we will focus on the use of magnetic tweezers to study the behavior of enzymes involved in DNA replication. Depending on the DNA substrate used, magnetic tweezers give access either to the advancement in real time of the so-called replication fork or to the torsional state (the so-called supercoiled density) of the DNA molecule. We will show how the new tools at our disposal can be used to gain an unprecedented description of the kinetic properties of enzymes. The comparison of these results with theoretical models allows us to get insight into the mechanism used by the enzymes under study. This analysis is often out of reach of more classical, bulk techniques.

[1]  B. Alberts,et al.  Effects of the bacteriophage T4 gene 41 and gene 32 proteins on RNA primer synthesis: coupling of leading- and lagging-strand DNA synthesis at a replication fork. , 1990, Biochemistry.

[2]  T. Lionnet,et al.  Single-molecule assay reveals strand switching and enhanced processivity of UvrD. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  M. Peliti,et al.  Stretching single stranded DNA, a model polyelectrolyte. , 2002, Physical review letters.

[4]  Daniel J. Müller,et al.  Atomic force microscopy: A forceful way with single molecules , 1999, Current Biology.

[5]  B. Alberts,et al.  Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus. , 1981, The Journal of biological chemistry.

[6]  J. Liphardt,et al.  Reversible Unfolding of Single RNA Molecules by Mechanical Force , 2001, Science.

[7]  R. Lavery,et al.  DNA: An Extensible Molecule , 1996, Science.

[8]  S. Benkovic,et al.  Accessory proteins function as matchmakers in the assembly of the T4 DNA polymerase holoenzyme , 1995, Current Biology.

[9]  T. Strick,et al.  Behavior of supercoiled DNA. , 1998, Biophysical journal.

[10]  S. Chu,et al.  Laser Manipulation of Atoms and Particles , 1991, Science.

[11]  Michelle D. Wang,et al.  Estimating the persistence length of a worm-like chain molecule from force-extension measurements. , 1999, Biophysical journal.

[12]  B. Alberts,et al.  Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical regions in a single-stranded DNA template. , 1981, The Journal of biological chemistry.

[13]  Kiwamu Saito,et al.  Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution , 1995, Nature.

[14]  R Lavery,et al.  Stretched and overwound DNA forms a Pauling-like structure with exposed bases. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  N. Nossal,et al.  Characterization of the bacteriophage T4 gene 41 DNA helicase. , 1989, The Journal of biological chemistry.

[16]  C. Schmidt,et al.  Signals and noise in micromechanical measurements. , 1998, Methods in cell biology.

[17]  S. Benkovic,et al.  RNA Primer Handoff in Bacteriophage T4 DNA Replication , 2008, Journal of Biological Chemistry.

[18]  T. Strick,et al.  Twisting and stretching single DNA molecules. , 2000, Progress in biophysics and molecular biology.

[19]  D. Hinton,et al.  Bacteriophage T4 DNA primase-helicase. Characterization of oligomer synthesis by T4 61 protein alone and in conjunction with T4 41 protein. , 1987, The Journal of biological chemistry.

[20]  Gilles Charvin,et al.  Stretching of macromolecules and proteins , 2003 .

[21]  S. Benkovic,et al.  Stoichiometry and DNA Unwinding by the Bacteriophage T4 41:59 Helicase* , 1996, The Journal of Biological Chemistry.

[22]  S. Benkovic,et al.  The Oligomeric T4 Primase Is the Functional Form during Replication* , 2005, Journal of Biological Chemistry.

[23]  John B. Shoven,et al.  I , Edinburgh Medical and Surgical Journal.

[24]  E. Siggia,et al.  Driving proteins off DNA using applied tension. , 1997, Biophysical journal.

[25]  S. Weiss.  Fluorescence spectroscopy of single biomolecules. , 1999, Science.

[26]  E. Evans,et al.  Dynamic strength of molecular adhesion bonds. , 1997, Biophysical journal.

[27]  J. Griffith,et al.  Architecture of the Replication Complex and DNA Loops at the Fork Generated by the Bacteriophage T4 Proteins* , 2003, Journal of Biological Chemistry.

[28]  Anindya Dutta,et al.  DNA replication in eukaryotic cells. , 2002, Annual review of biochemistry.

[29]  Marko,et al.  Statistical mechanics of supercoiled DNA. , 1995, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[30]  S. Benkovic,et al.  Replisome-mediated DNA replication. , 2001, Annual review of biochemistry.

[31]  S. Smith,et al.  Single-molecule studies of DNA mechanics. , 2000, Current opinion in structural biology.

[32]  David Bensimon,et al.  Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[34]  B. Alberts,et al.  Purification and characterization of bacteriophage T4 gene 59 protein. A DNA helicase assembly protein involved in DNA replication. , 1994, The Journal of biological chemistry.

[35]  D. Hinton,et al.  Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein. , 1987, The Journal of biological chemistry.

[36]  S. Smith,et al.  Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. , 1992, Science.

[37]  B. Alberts,et al.  Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[38]  S. Benkovic,et al.  Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism , 2007, Proceedings of the National Academy of Sciences.