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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

Assembly of bacterial microchamber.A) Picture of an assembled microchamber connected to a reservoir at one side and to a needle at the other side, which will be connected later to a syringe pump. B) Schematic of a cross section of the microchamber. C) Image of bacteria growing in 2 dimensions.
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pone.0134892.g001: Assembly of bacterial microchamber.A) Picture of an assembled microchamber connected to a reservoir at one side and to a needle at the other side, which will be connected later to a syringe pump. B) Schematic of a cross section of the microchamber. C) Image of bacteria growing in 2 dimensions.

Mentions: A PDMS chamber is obtained by mixing reagents from the Sylgard 184 Silicone Elastomer Kit as specified from the manufacturer (10:1 elastomer: curing agent). The silicon mix is poured onto a microchamber mold and incubated at 100°C for 45 min to 1 hr. Microchambers are then removed from the mold and covered with a glass coverslip previously incubated in plasma cleaner for 5 min to clean the surface. A 1–2% gel pad is prepared by mixing M9 glucose media with agarose. When melted, ~500 μl of this M9-agarose gel is poured on a mold to obtain an agarose gel pad 400 μm thick. 5 μl of a 1/100 dilution in fresh M9 glucose of an overnight bacterial culture at 30°C are placed on the clean glass coverslip. The drop of bacterial suspension is covered with the agarose gel pad and incubated for 3–5 min at room temperature (RT). During this time, silicon glue (elastosil E41 from Wacker) is applied on the PDMS chamber which is clamped on the assembled glass/bacteria/gel pad and is kept at RT for 40 min-1 hr. After this time, using a needle one side of the microchamber is connected to a reservoir, which is used to introduce the M9 0.4% glucose, 0.006% casamino acids, media (Fig 1). The other side of the microchamber is connected to a syringe pump, which is used to flow the media between the PDMS microchamber and the gel pad. The M9 glucose media reaches the bacteria by simple diffusion through the agarose gel pad. The microscope is equipped with a thermo-regulated sample holder. Cells are placed on the microscope stage at 30°C for 90–120 min and observed to ensure growth and to take control pictures of growing cells. Fluorescence images of group of bacteria are then taken every 1 to 2 min for 40 min.


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)

Assembly of bacterial microchamber.A) Picture of an assembled microchamber connected to a reservoir at one side and to a needle at the other side, which will be connected later to a syringe pump. B) Schematic of a cross section of the microchamber. C) Image of bacteria growing in 2 dimensions.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134892.g001: Assembly of bacterial microchamber.A) Picture of an assembled microchamber connected to a reservoir at one side and to a needle at the other side, which will be connected later to a syringe pump. B) Schematic of a cross section of the microchamber. C) Image of bacteria growing in 2 dimensions.
Mentions: A PDMS chamber is obtained by mixing reagents from the Sylgard 184 Silicone Elastomer Kit as specified from the manufacturer (10:1 elastomer: curing agent). The silicon mix is poured onto a microchamber mold and incubated at 100°C for 45 min to 1 hr. Microchambers are then removed from the mold and covered with a glass coverslip previously incubated in plasma cleaner for 5 min to clean the surface. A 1–2% gel pad is prepared by mixing M9 glucose media with agarose. When melted, ~500 μl of this M9-agarose gel is poured on a mold to obtain an agarose gel pad 400 μm thick. 5 μl of a 1/100 dilution in fresh M9 glucose of an overnight bacterial culture at 30°C are placed on the clean glass coverslip. The drop of bacterial suspension is covered with the agarose gel pad and incubated for 3–5 min at room temperature (RT). During this time, silicon glue (elastosil E41 from Wacker) is applied on the PDMS chamber which is clamped on the assembled glass/bacteria/gel pad and is kept at RT for 40 min-1 hr. After this time, using a needle one side of the microchamber is connected to a reservoir, which is used to introduce the M9 0.4% glucose, 0.006% casamino acids, media (Fig 1). The other side of the microchamber is connected to a syringe pump, which is used to flow the media between the PDMS microchamber and the gel pad. The M9 glucose media reaches the bacteria by simple diffusion through the agarose gel pad. The microscope is equipped with a thermo-regulated sample holder. Cells are placed on the microscope stage at 30°C for 90–120 min and observed to ensure growth and to take control pictures of growing cells. Fluorescence images of group of bacteria are then taken every 1 to 2 min for 40 min.

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