JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System

Published: August 21st, 2016

DOI:

10.3791/54434

1School of Environmental and Life Sciences, University of Newcastle

We describe here a system utilizing a site-specific, reversible in vivo protein block to stall and collapse replication forks in Escherichia coli. The establishment of the replication block is evaluated by fluorescence microscopy and neutral-neutral 2-dimensional agarose gel electrophoresis is used to visualize replication intermediates.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

During DNA replication, the replisome faces obstacles on the DNA that impair its progression. DNA damage including lesions and gaps as well as aberrant structures can prevent the replisome from proceeding1. Recently, it has been found that proteins bound to the DNA are the most common source of impediment to replication fork progression2. The knowledge of the events following the encounter of the replisome with a nucleoprotein block has previously been limited by the inability to induce such a block in the chromosome of a living cell at a known location. In vitro analysis has enhanced our understanding of the kinetic behavior of an activ....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

1. Blocking Replication with FROS

  1. Inducing the Replication Blockage
    1. Dilute a fresh overnight culture of an E. coli strain carrying a tetO array and pKM118 to OD600nm = 0.01 in a dilute complex medium (0.1% tryptone, 0.05% yeast extract, 0.1% NaCl, 0.17 M KH2PO4, 0.72 M K2HPO4) with antibiotics as required for selection.
      Note: Do not add tetracycline for selection as it is not compatible with this system. Ma.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

The FROS is an inducible, site-specific nucleoprotein block that enables replication intermediates to be visualized in living cells12,18. A general experimental design for sampling cells is illustrated in Figure 1. The timing of the sampling and variations in genetic background make this a versatile system for studying the repair of such a block. The schematic illustrates how temperature sensitive mutants, such as dnaBts and dnaCts that have b.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

During chromosome duplication, the replication machinery will encounter various impediments that prevent its progress. To ensure the entire single-origin chromosome is replicated, bacteria have numerous pathways for repair of the DNA that then enables the replisome to be reloaded20,21. Lesions, single stranded breaks, double stranded breaks and proteins tightly bound to the DNA may each be dealt with using a dedicated pathway, although there is likely to be significant overlap in these pathways. The most commo.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was supported by the Australian Research Council [DP11010246].

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
Tryptone Sigma-Aldrich 16922 Growth media component
Sodium Chloride VWR 27810.364 Growth media component
Yeast extract Sigma-Aldrich 92144 Growth media component
Potassium phosphate monobasic Sigma-Aldrich P9791 Growth media component; Potassium buffer component
Potassium phosphate dibasic Sigma-Aldrich P3786 Growth media component; Potassium buffer component
L-Arabinose Sigma-Aldrich A3256 For induction of TetR-YFP production
Anhydrotetracycline hydrochloride Sigma-Aldrich 37919 Release of replication bloackage
Axioskop 2 Fluorescence microscope Zeiss 452310 Visualization of cells
eYFP filter set Chroma Technology 41028 Visualization of YFP
CCD camera Hamamatsu Orca-AG Visualization of cells
MetaMorph  Software (Molecular Devices) SDR Scientific 31282 Version 7.8.0.0 used in the preparation of this manuscript
Agarose Bioline BIO-41025 For agarose plugs and gel electrophoresis
Original Glass Water Repellent (Rain-X) Autobarn DIO1470 For agarose plug manufacture
TRIS VWR VWRC103157P TE, TBE buffer component
Ethylene diaminetetraacetic acid Ajax Finechem AJA180 0.5 M EDTA disodium salt solution adjusted to pH 8.0 with NaOH.
Sodium azide Sigma-Aldrich S2002 Bacteriostatic agent
Hydrochloric acid Sigma-Aldrich 258148 TE buffer component
Sodium deoxycholate Sigma-Aldrich D6750 Cell lysis buffer component
N-Lauroylsarcosine sodium salt (Sarkosyl) Sigma-Aldrich L5125 Cell lysis and ESP buffer component
Rnase A Sigma-Aldrich R6513 Cell lysis buffer component
Lysozyme Amresco 6300 Cell lysis buffer component
Proteinase K Amresco AM0706 ESP buffer component
Sub-Cell Model 192 Cell BioRad 1704507 Electrophoresis system
UV transilluminator 2000 BioRad 1708110 Visualization of DNA
Ethidium Bromide BioRad 1610433 Visualization of DNA
Boric acid VWR PROL20185.360 TBE component
Hybond-XL nylon memrbane Amersham RPN203S Zeta-Probe Memrbane (BioRad 1620159) can also be used
3MM Whatman chromatography paper GE Healthcare Life Sciences 3030690 Southern blotting
HL-2000 Hybrilinker UVP 95-0031-01/02 Crosslinking of DNA and hybridization
Deoxyribonucleic acid from salmon sperm Sigma-Aldrich 31149 Hybridization buffer component
Sodium hydroxide Sigma-Aldrich S5881 Denaturation buffer component
Trisodium citrate dihydrate VWR PROL27833.363 Transfer buffer
Sodium dodecyl sulphate (SDS) Amresco 227 Wash buffer component
Bovine serum albumin Sigma-Aldrich A7906 Hybridization buffer component
Random Hexamer Primers Bioline BIO-38028
Klenow fragment New England BioLabs M0212L
dNTP Set Bioline BIO-39025
Adenosine 5’-triphosphate-32P-ATP PerkinElmer BLU502A
Storage Phosphor Screen GE Healthcare Life Sciences GEHE28-9564-76 BAS-IP MS 3543 E multipurpose standard 35x43cm screen
Typhoon FLA 7000 GE Healthcare Life Sciences 28-9558-09 Visualization of blot
Hybridization bottle UVP 07-0194-02 35 x 300mm

  1. Mirkin, E. V., Mirkin, S. M. Replication fork stalling at natural impediments. Microbiol Mol Biol Rev. 71 (1), 13-35 (2007).
  2. Gupta, M. K., et al. Protein-DNA complexes are the primary sources of replication fork pausing in Escherichia coli. Proc Natl Acad Sci U S A. 110 (18), 7252-7257 (2013).
  3. McGlynn, P., Guy, C. P. Replication forks blocked by protein-DNA complexes have limited stability in vitro. J Mol Biol. 381 (2), 249-255 (2008).
  4. Tanner, N. A., et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat Struct Mol Biol. 15 (2), 170-176 (2008).
  5. Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., van Oijen, A. M. Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature. 457 (7227), 336-339 (2009).
  6. Rudolph, C. J., Upton, A. L., Lloyd, R. G. Replication fork stalling and cell cycle arrest in UV-irradiated Escherichia coli. Genes Dev. 21 (6), 668-681 (2007).
  7. Jeiranian, H. A., Courcelle, C. T., Courcelle, J. Inefficient replication reduces RecA-mediated repair of UV-damaged plasmids introduced into competent Escherichia coli. Plasmid. 68 (2), 113-124 (2012).
  8. Le Masson, M., Baharoglu, Z., Michel, B. ruvA and ruvB mutants specifically impaired for replication fork reversal. Mol Microbiol. 70 (2), 537-548 (2008).
  9. Wang, X., Liu, X., Possoz, C., Sherratt, D. J. The two Escherichia coli chromosome arms locate to separate cell halves. Genes Dev. 20 (13), 1727-1731 (2006).
  10. Lau, I. F., et al. Spatial and temporal organization of replicating Escherichia coli chromosomes. Mol Microbiol. 49 (3), 731-743 (2003).
  11. Gordon, G. S., et al. Chromosome and low copy plasmid segregation in E. coli: visual evidence for distinct mechanisms. Cell. 90 (6), 1113-1121 (1997).
  12. Possoz, C., Filipe, S. R., Grainge, I., Sherratt, D. J. Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo. EMBO J. 25 (11), 2596-2604 (2006).
  13. Payne, B. T., et al. Replication fork blockage by transcription factor-DNA complexes in Escherichia coli. Nucleic Acids Res. 34 (18), 5194-5202 (2006).
  14. Brewer, B. J., Fangman, W. L. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell. 51 (3), 463-471 (1987).
  15. Jeiranian, H. A., Schalow, B. J., Courcelle, J. Visualization of UV-induced replication intermediates in E. coli using two-dimensional agarose-gel analysis. J Vis Exp. (46), (2010).
  16. Southern, E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 98 (3), 503-517 (1975).
  17. Courcelle, J., Donaldson, J. R., Chow, K. H., Courcelle, C. T. DNA damage-induced replication fork regression and processing in Escherichia coli. Science. 299 (5609), 1064-1067 (2003).
  18. Mettrick, K. A., Grainge, I. Stability of blocked replication forks in vivo. Nucleic Acids Res. , (2015).
  19. Church, G. M., Gilbert, W. Genomic sequencing. Proc Natl Acad Sci U S A. 81 (7), 1991-1995 (1984).
  20. Michel, B., Grompone, G., Flores, M. J., Bidnenko, V. Multiple pathways process stalled replication forks. Proc Natl Acad Sci U S A. 101 (35), 12783-12788 (2004).
  21. Michel, B., Boubakri, H., Baharoglu, Z., LeMasson, M., Lestini, R. Recombination proteins and rescue of arrested replication forks. DNA Repair (Amst). 6 (7), 967-980 (2007).
  22. Carl, P. L. Escherichia coli mutants with temperature-sensitive synthesis of DNA. Mol Gen Genet. 109 (2), 107-122 (1970).
  23. Nawotka, K. A., Huberman, J. A. Two-dimensional gel electrophoretic method for mapping DNA replicons. Mol Cell Biol. 8 (4), 1408-1413 (1988).
  24. Cole, R. S., Levitan, D., Sinden, R. R. Removal of psoralen interstrand cross-links from DNA of Escherichia coli: mechanism and genetic control. J Mol Biol. 103 (1), 39-59 (1976).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved