Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

[1]  A. Kornberg,et al.  Complete replication of templates by Escherichia coli DNA polymerase III holoenzyme. , 1985, The Journal of biological chemistry.

[2]  C. Richardson,et al.  Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. , 1987, The Journal of biological chemistry.

[3]  M. O’Donnell,et al.  Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme. , 1990, The Journal of biological chemistry.

[4]  C. Richardson,et al.  A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[5]  K. Marians.  Phi X174-type primosomal proteins: purification and assay. , 1995, Methods in enzymology.

[6]  B. Åkerman,et al.  Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO. , 1996, Nucleic acids research.

[7]  J. Griffith,et al.  Coordinated leading and lagging strand DNA synthesis on a minicircular template. , 1998, Molecular cell.

[8]  Premnath,et al.  Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption. , 1998, Macromolecules.

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

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

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

[12]  V. Demidov,et al.  Rolling-circle amplification in DNA diagnostics: the power of simplicity , 2002, Expert review of molecular diagnostics.

[13]  Taekjip Ha,et al.  Structural dynamics and processing of nucleic acids revealed by single-molecule spectroscopy. , 2004, Biochemistry.

[14]  Charles R Cantor,et al.  Real-time monitoring of branched rolling-circle DNA amplification with peptide nucleic acid beacon. , 2004, Analytical biochemistry.

[15]  C. Anselmi,et al.  Nanoscale mechanical and dynamical properties of DNA single molecules. , 2005, Biophysical chemistry.

[16]  N. Cozzarelli,et al.  Independence of replisomes in Escherichia coli chromosomal replication. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. O’Donnell,et al.  Cellular DNA replicases: components and dynamics at the replication fork. , 2005, Annual review of biochemistry.

[18]  Boriana Marintcheva,et al.  A unique loop in T7 DNA polymerase mediates the binding of helicase-primase, DNA binding protein, and processivity factor. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[19]  N. Broude,et al.  High-density fluorescently labeled rolling-circle amplicons for DNA diagnostics. , 2005, Analytical biochemistry.

[20]  X. Xie,et al.  DNA primase acts as a molecular brake in DNA replication , 2006, Nature.

[21]  R. Heller,et al.  Replication fork reactivation downstream of a blocked nascent leading strand , 2006, Nature.

[22]  Antoine M. van Oijen,et al.  A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[23]  C. Dekker,et al.  Single-molecule studies of nucleic acid motors. , 2007, Current opinion in structural biology.

[24]  R. Lange,et al.  The targets of currently used antibacterial agents: lessons for drug discovery. , 2007, Current pharmaceutical design.

[25]  M. O’Donnell,et al.  Replisome mechanics: insights into a twin DNA polymerase machine. , 2007, Trends in microbiology.

[26]  N. Tanner,et al.  Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement. , 2007, Molecular cell.

[27]  Gijs J. L. Wuite,et al.  See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins , 2008, Nucleic acids research.

[28]  Single-molecule studies of fork dynamics in Escherichia coli DNA replication. , 2008, Nature structural & molecular biology.

[29]  N. Tanner,et al.  Single-molecule studies of fork dynamics in Escherichia coli DNA replication , 2008, Nature Structural &Molecular Biology.

[30]  A. Berdis.  DNA polymerases as therapeutic targets. , 2008, Biochemistry.

[31]  Visualization of long human telomere mimics by single-molecule fluorescence imaging. , 2008, The journal of physical chemistry. B.

[32]  A. Pyle.  Translocation and unwinding mechanisms of RNA and DNA helicases. , 2008, Annual review of biophysics.

[33]  J. Loparo,et al.  Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis , 2009, Nature.