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Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

Bentchikou E, Chagneau C, Long E, Matelot M, Allemand JF, Michel B - PLoS ONE (2015)

Bottom Line: In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB).A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used.Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, Gif sur Yvette, France.

ABSTRACT
In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.

No MeSH data available.


Related in: MedlinePlus

Examples of stable and unstable RecO-Venus and RecG-Venus foci, and of stable and unstable foci in cells that express Venus from the recG promoter.For each bacteria, the first four frames of the fluorescent series of images are shown below the brightfield picture (top). See the legend to Fig 2 for details.
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pone.0134892.g003: Examples of stable and unstable RecO-Venus and RecG-Venus foci, and of stable and unstable foci in cells that express Venus from the recG promoter.For each bacteria, the first four frames of the fluorescent series of images are shown below the brightfield picture (top). See the legend to Fig 2 for details.

Mentions: Fluorescence images were first treated with a custom-made Matlab program to compensate unequal illumination assuming a Gaussian profile illumination. Image treatments and analysis were then done using the open-source software ImageJ. Images were treated to remove background and thus allow better foci detection (S3 Fig). For this purpose, fluorescence images were duplicated and treated using the ImageJ’s plugin Filter “Sigma Filter Plus”. This plugin-Filter provides a selective mean (averaging) filter. The filter smooths an image by taking an average over the neighbouring pixels, but only includes those pixels that have a value not deviating from the current pixel by more than a given range. One of the duplicated images was treated using the plugin filter parameters: Radius = 10 pixels, using pixels within 5 sigma range and Minimum Pixel Fraction = 0.2. The other fluorescent image was not treated, and the two images were subtracted to create an image with only the fluorescence spots (Figs 1C, 2, 3 and 4, and S3 Fig). Foci were identified manually as groups of at least 5 pixels, Their position coordinates were determined by finding the position of spot fluorescence intensity maxima. A line was drawn on each bacterium on the brightfield image from pole to pole and translated on the treated fluorescent images. This line was used as a fixed mark to compare foci position, defined by the position of the maximum intensity pixel(s), in the four analyzed frames. Additionally, for foci that were centered in the longitudinal axis, intensity along the line was used to determine their stability (S4 Fig). The first four frames (800 ms) were analyzed; foci were called stable when they remained immobile or moved less than one pixel for at least 3 frames (authorizing one blinking event) and called unstable when they moved for more than 1 pixels or disappeared (see examples in Figs 2, 3 and 4, and S4 Fig). Foci were called central when located between 0.4 and 0.5, and lateral when located between 0.15 and 0.33 of cell length. Nearly no foci were polar (<0.15) and foci between 0.33 and 0.4 were considered as neither central nor lateral.


Are the SSB-Interacting Proteins RecO, RecG, PriA and the DnaB-Interacting Protein Rep Bound to Progressing Replication Forks in Escherichia coli?

Bentchikou E, Chagneau C, Long E, Matelot M, Allemand JF, Michel B - PLoS ONE (2015)

Examples of stable and unstable RecO-Venus and RecG-Venus foci, and of stable and unstable foci in cells that express Venus from the recG promoter.For each bacteria, the first four frames of the fluorescent series of images are shown below the brightfield picture (top). See the legend to Fig 2 for details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526528&req=5

pone.0134892.g003: Examples of stable and unstable RecO-Venus and RecG-Venus foci, and of stable and unstable foci in cells that express Venus from the recG promoter.For each bacteria, the first four frames of the fluorescent series of images are shown below the brightfield picture (top). See the legend to Fig 2 for details.
Mentions: Fluorescence images were first treated with a custom-made Matlab program to compensate unequal illumination assuming a Gaussian profile illumination. Image treatments and analysis were then done using the open-source software ImageJ. Images were treated to remove background and thus allow better foci detection (S3 Fig). For this purpose, fluorescence images were duplicated and treated using the ImageJ’s plugin Filter “Sigma Filter Plus”. This plugin-Filter provides a selective mean (averaging) filter. The filter smooths an image by taking an average over the neighbouring pixels, but only includes those pixels that have a value not deviating from the current pixel by more than a given range. One of the duplicated images was treated using the plugin filter parameters: Radius = 10 pixels, using pixels within 5 sigma range and Minimum Pixel Fraction = 0.2. The other fluorescent image was not treated, and the two images were subtracted to create an image with only the fluorescence spots (Figs 1C, 2, 3 and 4, and S3 Fig). Foci were identified manually as groups of at least 5 pixels, Their position coordinates were determined by finding the position of spot fluorescence intensity maxima. A line was drawn on each bacterium on the brightfield image from pole to pole and translated on the treated fluorescent images. This line was used as a fixed mark to compare foci position, defined by the position of the maximum intensity pixel(s), in the four analyzed frames. Additionally, for foci that were centered in the longitudinal axis, intensity along the line was used to determine their stability (S4 Fig). The first four frames (800 ms) were analyzed; foci were called stable when they remained immobile or moved less than one pixel for at least 3 frames (authorizing one blinking event) and called unstable when they moved for more than 1 pixels or disappeared (see examples in Figs 2, 3 and 4, and S4 Fig). Foci were called central when located between 0.4 and 0.5, and lateral when located between 0.15 and 0.33 of cell length. Nearly no foci were polar (<0.15) and foci between 0.33 and 0.4 were considered as neither central nor lateral.

Bottom Line: In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB).A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used.Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris Sud, Gif sur Yvette, France.

ABSTRACT
In all organisms several enzymes that are needed upon replication impediment are targeted to replication forks by interaction with a replication protein. In most cases these proteins interact with the polymerase clamp or with single-stranded DNA binding proteins (SSB). In Escherichia coli an accessory replicative helicase was also shown to interact with the DnaB replicative helicase. Here we have used cytological observation of Venus fluorescent fusion proteins expressed from their endogenous loci in live E. coli cells to determine whether DNA repair and replication restart proteins that interact with a replication protein travel with replication forks. A custom-made microscope that detects active replisome molecules provided that they are present in at least three copies was used. Neither the recombination proteins RecO and RecG, nor the replication accessory helicase Rep are detected specifically in replicating cells in our assay, indicating that either they are not present at progressing replication forks or they are present in less than three copies. The Venus-PriA fusion protein formed foci even in the absence of replication forks, which prevented us from reaching a conclusion.

No MeSH data available.


Related in: MedlinePlus