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 active replisome when it meets a nucleoprotein blockage3, as well as the mechanistic details of the replisome itself4,5. Current understanding of the repair of replication is generally undertaken with UV as the damaging agent and studied using plasmid DNA in vivo6-8. While the proteins that may be involved in repair of DNA after it encounters an in vivo nucleoprotein block are generally understood from these studies, whether there are variations in the molecular events within the repair pathways owing to the distinct cause in the replication block is still yet to be determined.
Here, we describe a system that allows a nucleoprotein block to be established in a specific location of the chromosome using a Fluorescent Repressor Operator System (FROS). We utilize a strain of E. coli that has had an array of 240 tetO sites incorporated into the chromosome9. Each tetO site within the array has a 10 bp random sequence flanking it to increase the stability of the array by preventing RecA-mediated recombination within the array. This array, and variations of it, were originally used to understand E. coli chromosome dynamics10,11 but were then adapted to prevent in vivo replication12. The array has been found to be stably maintained and to block close to 100% of replication forks when bound by TetR10,12. The use of the similar lacO array in vitro has found as few as 22 sites were sufficient to block 90% of replication, although this shorter array was less effective in vivo13. To adapt the array to create a nucleoprotein blockage, the repressor protein must be highly overproduced under optimized conditions where it then binds to the array to create a roadblock. The formation of the blockage, and its subsequent release, can be monitored through the use of fluorescence microscopy if a fluorescently tagged variant of the Tet repressor is used. The status of replication in each cell is indicated by the number of foci seen, where a single focal point means only one copy of the array is present within the cell and multiple foci are indicative of active replication. This active replication is enabled when the nucleoprotein blockage is reversed by the addition of the gratuitous inducer that decreases the binding affinity of TetR for the operator site sufficiently for the replisome to proceed through the array. The repressor protein is still able to bind to the DNA with high enough affinity that the now multiple copies of the array can be visualized.
More intricate details of the events at the nucleoprotein blockage can be discovered using neutral-neutral two-dimensional agarose gel electrophoresis and Southern hybridization14-16. These techniques allow the analysis of DNA structures across the population. The replication intermediates that are formed during the event, and potentially remain unrepaired, can be visualized. By varying the restriction enzyme and probe utilized, the intermediates can be visualized not only in the array region but also upstream of the array when the replication fork regresses17,18. The regression takes place subsequent to the replisome dissociation; the leading and lagging nascent strands separate from the template strands and anneal to each other as the template strands concurrently re-anneal resulting in a four-way DNA structure (a Holliday junction).
Using this system it has been shown that the replication fork is not stable when it encounters this block18. In addition, temperature sensitive derivatives of replisome components can be utilized to prevent reloading of the replication fork once it has collapsed. Once a block is established, the strain can be shifted to a non-permissive temperature to ensure a synchronous deactivation of the replisome and a controlled prevention of reloading. This temperature-induced deactivation ensures all of the forks within the population have collapsed at a given time and allows the assessment of what happens when the replisome collapses, how the DNA is processed, and what is required to restart the process of DNA replication.
An advantage of the system described here is that the nucleoprotein block is fully reversible; therefore, the ability of the cells to recover from the nucleoprotein block is able to be followed. The addition of anhydrotetracycline to the cells will relieve the tight binding of the repressor, allowing a replication fork to proceed through and the cell to regain viability. The relief of the blockage can be visualized by neutral-neutral two-dimensional agarose gel electrophoresis after 5 min, and by microscopy within 10 min. Furthermore, viability analysis can reveal the ability of the strain to recover from the replication blockage and continue to proliferate.
By altering the genetic background of the strains used in the experimental procedure described here, the repair pathways for this type of blockage can be elucidated.
1. Blocking Replication with FROS
Figure 1: Overview of the FROS Replication Block and Release Experimental Procedure. E. coli strains carrying the tetO array are grown at 30 °C. When the cells reach an OD600nm of above 0.05, TetR-YFP production is induced with arabinose (ara; 0.1%). Continue to grow a subpopulation of uninduced cells to act as controls at 30 °C. A sample of the induced cells is analyzed via fluorescence microscopy after 1 – 2 hr (see 2.1). If replication is confirmed to be blocked, samples are taken for 2-D gel analysis indicated by the test tubes (see 3.1) and for viability tests (optional). The replication blockage can be removed with anhydrotetracycline (AT; 0.1 µg/ml) and cells analyzed 10 min later. If cells carry a temperature sensitive allele (e.g. dnaBts), this can be inactivated by shifting the blocked cells to 42 °C for appropriate analysis. Please click here to view a larger version of this figure.
2. Fluorescence Microscopy
3. DNA Extraction/Agarose Plugs
4. Neutral-neutral 2-Dimensional Gel Electrophoresis
5. Southern Hybridization
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 been used previously18, can be utilized in this system.
The induction of the FROS was carried out in an E. coli strain as shown in Figure 1 for non-temperature sensitive strains. To confirm that a nucleoprotein block had been established, cells were visualized using fluorescence microscopy after 1 hr of growth in 0.1% arabinose. This timeframe is usually adequate to produce the block in a wild type strain but more fastidious strains may require optimization. The majority of the cells contained 1 focus (Figure 2A, + ara), corresponding to only one copy of the array within the cell thereby indicating replication had been blocked. The numbers of cells with 1 focus, 2 foci or more than 2 foci were counted in a population of more than 100 cells. 92% of cells were found to have 1 focus with the remaining 8% with 2 foci. It was presumed that cells that had two foci spaced well apart, representing two copies of the array, would have the next round of replication blocked. Cells with two foci close together signify that the array has recently been replicated and the nucleoprotein block has not yet been achieved.
The nucleoprotein block was reversed by the addition of anhydrotetracycline (AT) and subsequent duplication of the array visualized as the accumulation of cells with multiple foci (Figure 2A, + ara/AT). Multiple foci within a cell visualized by fluorescence microcopy signify the array has been successfully replicated and sufficient time has passed to allow the loci to move apart, overcoming any sister chromosome cohesion that was present. In this instance, 10 minutes after the addition of AT, 94% of cells had 2 or more foci and up to 8 foci per cell were visualized.
To ensure that the nucleoprotein block did indeed inhibit replication, cell viability was analyzed (Figure 2B). Cells that have had replication inhibited by the FROS show an approximately 1000 fold decrease in colony forming units compared to cells that were not grown in the presence of arabinose. Cells that were subsequently grown in the presence of anhydrotetracycline show reversal of this growth inhibition. If the cells were unable to repair the DNA after release of the nucleoprotein blockage, a reduction in viability would result.
To visualize the replication intermediates, cells can be lysed within agarose plugs and the protein and RNA removed. The DNA can then be digested with an appropriate restriction enzyme for the region of interest. DNA pictured in Figure 3A was digested with EcoRV which cuts the DNA immediately before the array, within the array and after the array to give 5.5 kb and 6.7 kb fragments in the region of interest (Figure 3B). Fragments are ideally ~3-7 kb for the protocol described here. Fragments outside this range may require alteration of the agarose concentrations used and the electrophoresis conditions to achieve good separation. The DNA was electrophoresed in the first dimension and visualized (Figure 3A). If the DNA has digested completely, all lanes will have run evenly and there will be a high amount of low molecular weight (less than approximately 3 kb, depending on the restriction enzyme). DNA present in the well suggests incomplete cell lysis and a lot of high molecular weight DNA (above 10 kb) implies incomplete DNA digestion.
The DNA of interest in each lane was excised. In Figure 3A, DNA above 5 kb was included. The subsequent second dimension of the gel was electrophoresed, resulting in a diagonal of linear DNA (Figure 3C). The array DNA within this gel was then visualized by Southern hybridization (Figure 3D). In the unblocked (- ara sample), two spots can be seen, corresponding to the 5.5 kb and 6.7 kb fragments of the array, as expected. The sample which had replication blocked (+ ara) showed a decrease in the 5.5 kb spot in comparison and the addition of an elliptical signal, indicating an accumulation of Y-shaped DNA in the sample. The signal is localized to one position, signifying the replication forks are arresting in a similar place throughout the population. On addition of anhydrotetracyline, the Y-shaped DNA signal can no longer be seen. A recent restart is indicated by the presence of a signal of the entire Y-arc, depicting the forks migrating through the region.
It is important to normalize the DNA within each agarose plug to ensure the signals of the samples are even. The signals can be quantified with appropriate imaging software. Another common intermediate seen is that indicating Holliday junction (HJ) formation. A HJ is visualized as a cone signal at the top of the Y-arc and a spike from the linear DNA at the end of the Y arc (Figure 3D).
Figure 2: Fluorescence Microscopy and Viability. (A) An E. coli strain carrying the tetO array was grown at 30 °C in the presence of 0.1% arabinose (+ ara) for 1 hr and then anhydrotetracycline (AT) was added for 10 min to a subpopulation to release the replication block. Representative micrographs are shown for YFP production (top panel). Scale bar = 2 µm. The proportion of cells carrying 1, 2 or more than 2 foci were enumerated and are presented as a percentage (lower panel). (B) Cells grown in the absence of arabinose (- ara), with arabinose (+ ara) and with both arabinose and anhydrotetracycline (AT; 10 min) were serial diluted 10-fold and either spotted or spread onto agar containing ampicillin only (-/+ ara samples) or ampicillin with anhydrotetracycline (+ AT sample) and grown at 30 °C to determine cell viability. Top: representative plate showing growth at indicated dilutions. Bottom: graph showing the average CFU/ml +/- SEM. Please click here to view a larger version of this figure.
Figure 3: Visualization of DNA Replication Intermediates at a Nucleoprotein Block. (A) Genomic DNA from cells (1, – ara; 2, + ara; 3, + ara/AT) that were lysed within agarose plugs and digested with EcoRV restriction enzyme was separated on a 0.4% agarose gel (55 V, 16 hr). 3 kb, 5 kb and 10 kb bands are indicated. The DNA above the 5 kb marker was excised as indicated by the white boxes. (B) Schematic of the EcoRV digest of the array region. Replication forks entering the array from the origin become blocked within the 5.5 kb fragment when the cells have been grown in the presence of arabinose. Restriction sites utilized are indicated by arrows and the array is shown as a box with lines. (C) The excised DNA was rotated 90° and separated in a 0.8% agarose gel (220 V, 4 hr). The white box indicates the excision of DNA after separation. (D) The array region was subsequently visualized by Southern blot analysis using a radioactive probe to the array region. Cells with a replication block at this position will have the signal corresponding to the 5.5 kb fragment located on the Y-arc. Please click here to view a larger version of this figure.
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 common impediment to replisomes in living cells is a nucleoprotein block2. However, two longstanding difficulties in studying the collision of a replisome with a nucleoprotein blockage have been how to localize the event to a known position on the chromosome and how to make the event frequent enough to allow detailed analysis. The majority of the knowledge of how E. coli copes with stalled/collapsed replication forks comes from the use of UV as a DNA damaging agent.
The FROS protocol described here is a novel way of creating a site-specific nucleoprotein roadblock, similar to what the replisome will meet when it encounters proteins tightly bound to the chromosome during replication. The induced blockage and controlled release of this block allows the stages of DNA processing to be studied in discrete steps. The array of tetO sites used to block replication has previously been utilized to visualize chromosome organization9,10. While the array can be placed anywhere in the chromosome, for the purpose of the array described here, it should be distant from both the origin and the terminus to avoid complications in interpreting the signals observed from second rounds of initiation at the origin or from the replication fork proceeding around the opposite replichore. By optimizing the overproduction of TetR with the use of the dilute complex medium (protocol 1), we have found that sufficient TetR is produced to create a complete replication blockage that is stably maintained, while limiting the number of new rounds of replication that are initiated. The incorporation of replisome temperature sensitive mutations22 into the system ensures all replisomes are deactivated simultaneously and uncontrolled replisome reloading is prevented.
Whilst the number of foci per cell are useful for knowing the cell’s replication status, the most enlightening data for DNA repair is from the analysis of the replication intermediates revealed by 2-D gel electrophoresis during an active replication blockage. Analysis of different genetic backgrounds can identify proteins involved in processing and restart of collapsed replication forks by the different patterns observed following the 2-D gel protocol. Further, intermediates from the processing of collapsed replication forks can be present at the array region or in the DNA upstream of the array, and can be visualized using 2-D gel electrophoresis. Creating duplicate agarose plugs (Protocol 3) has the advantage of allowing for digestion of the same sample with different restriction enzymes to be able to visualize replication intermediates in successive DNA regions. Therefore, the array region should be located in a position where appropriate restriction enzymes can be used to probe these regions of interest using Southern hybridization.
The most common reasons that this technique may be unsuccessful are having either too little DNA which produces faint signals compared to background, or incomplete digestion of the DNA within the plugs. This can be caused by having too much DNA in the agarose plugs or the carry-over of some inhibitor from the lysis/washing steps. The number of cells taken for lysis within the agarose plugs may need to be varied from the protocol described here if modifications such as growth media are changed. However, it is futile to continue with the hybridization if the DNA in the plugs has not digested to completion. A repeat treatment of ESP may be required to better prepare the DNA (protocol 3.6).
If the replication intermediates that are of interest are only transient, or are particularly rare within the cell, the DNA sample may need to be enriched for them, for example with the use of BND-cellulose which will bind short stretches of single stranded DNA, such as those found in replicating chromosomes23. Stabilization of replication intermediates by crosslinking with psoralen has also been used as a method of preserving DNA integrity24. When the intermediates are rare, it is important to optimize the probe and blocking of the membrane to prevent background signals from interfering. On completion of the blots the signals may be quantified with appropriate image processing software.
The protocol described here is an optimized system for creating a site-specific nucleoprotein blockage and analyzing replication intermediates when the replisome is unable to proceed through the blockage. With the establishment and optimization of this technique, the roles of various helicases and recombination proteins in processing the replication fork and in allowing the restart of replication can be identified. By varying the genetic background, the effect of the absence of these proteins can be analyzed. However, this technique is limited by requiring not only viable strains but strains that are also able to overproduce TetR at a rate that will create a stable nucleoprotein blockage.
The authors have nothing to disclose.
This work was supported by the Australian Research Council [DP11010246].
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 |