Limits...
The rotation-coupled sliding of EcoRV.

Dikić J, Menges C, Clarke S, Kokkinidis M, Pingoud A, Wende W, Desbiolles P - Nucleic Acids Res. (2012)

Bottom Line: We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6.Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D(1)), revealing a significant size dependency.The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Kastler Brossel, ENS, UPMC-Paris6, CNRS UMR 8552, 24 rue Lhomond, 75005 Paris, France. jasmina.dikic@lkb.ens.fr

ABSTRACT
It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE-DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D(1)), revealing a significant size dependency. To account for the dependence of D(1) on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.

Show MeSH

Related in: MedlinePlus

Dependence of the normalized friction coefficient ξ (rl)/ξ (0) of a single-labeled (Models 1–4) or double-labeled (Model 5) EcoRV on the radius rl of the label(s): (1) linear diffusion with a rigid linker, (2) rotational diffusion with a rigid linker, (3) linear diffusion with a flexible linker, (4) rotational diffusion with a flexible linker, (5) rotational diffusion with a rigid linker. EcoRV is presented schematically in magenta, DNA in green and the label in orange. Experimental normalized friction coefficients ξ (in red) are calculated using ξ (rl)/ξ (0) = DCy3B/D1, where DCy3B is the linear diffusion constant of EcoRV labeled with Cy3B, and D1 is the linear diffusion constant of a protein variant with one or two label(s) of radius rl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351190&req=5

gkr1309-F3: Dependence of the normalized friction coefficient ξ (rl)/ξ (0) of a single-labeled (Models 1–4) or double-labeled (Model 5) EcoRV on the radius rl of the label(s): (1) linear diffusion with a rigid linker, (2) rotational diffusion with a rigid linker, (3) linear diffusion with a flexible linker, (4) rotational diffusion with a flexible linker, (5) rotational diffusion with a rigid linker. EcoRV is presented schematically in magenta, DNA in green and the label in orange. Experimental normalized friction coefficients ξ (in red) are calculated using ξ (rl)/ξ (0) = DCy3B/D1, where DCy3B is the linear diffusion constant of EcoRV labeled with Cy3B, and D1 is the linear diffusion constant of a protein variant with one or two label(s) of radius rl.

Mentions: The diffusion constant D1 of the labeled enzymes was derived from the mean square displacement (MSD) calculated from enzyme trajectories longer than 20 successive frames (28 trajectories for QD605-PEG2-EcoRV, 24 trajectories for QD605-PEG11-EcoRV, 27 trajectories for QDEO6-EcoRV and 45 trajectories for EcoRV-scRM6). The main error in the estimation of D1 stems from the inaccuracy in determining the position of the DNA ends, and thus the DNA stretch rate. This rate is included in the calculation of the MSD for taking into account the effective translocation of the enzyme along the DNA. Assuming a localization accuracy of ∼120 nm for the end-to-end distance (∼2 µm), we obtained ΔD/D ∼ 0.15, which was translated into error bars on the MSD plots (Figure 3).


The rotation-coupled sliding of EcoRV.

Dikić J, Menges C, Clarke S, Kokkinidis M, Pingoud A, Wende W, Desbiolles P - Nucleic Acids Res. (2012)

Dependence of the normalized friction coefficient ξ (rl)/ξ (0) of a single-labeled (Models 1–4) or double-labeled (Model 5) EcoRV on the radius rl of the label(s): (1) linear diffusion with a rigid linker, (2) rotational diffusion with a rigid linker, (3) linear diffusion with a flexible linker, (4) rotational diffusion with a flexible linker, (5) rotational diffusion with a rigid linker. EcoRV is presented schematically in magenta, DNA in green and the label in orange. Experimental normalized friction coefficients ξ (in red) are calculated using ξ (rl)/ξ (0) = DCy3B/D1, where DCy3B is the linear diffusion constant of EcoRV labeled with Cy3B, and D1 is the linear diffusion constant of a protein variant with one or two label(s) of radius rl.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351190&req=5

gkr1309-F3: Dependence of the normalized friction coefficient ξ (rl)/ξ (0) of a single-labeled (Models 1–4) or double-labeled (Model 5) EcoRV on the radius rl of the label(s): (1) linear diffusion with a rigid linker, (2) rotational diffusion with a rigid linker, (3) linear diffusion with a flexible linker, (4) rotational diffusion with a flexible linker, (5) rotational diffusion with a rigid linker. EcoRV is presented schematically in magenta, DNA in green and the label in orange. Experimental normalized friction coefficients ξ (in red) are calculated using ξ (rl)/ξ (0) = DCy3B/D1, where DCy3B is the linear diffusion constant of EcoRV labeled with Cy3B, and D1 is the linear diffusion constant of a protein variant with one or two label(s) of radius rl.
Mentions: The diffusion constant D1 of the labeled enzymes was derived from the mean square displacement (MSD) calculated from enzyme trajectories longer than 20 successive frames (28 trajectories for QD605-PEG2-EcoRV, 24 trajectories for QD605-PEG11-EcoRV, 27 trajectories for QDEO6-EcoRV and 45 trajectories for EcoRV-scRM6). The main error in the estimation of D1 stems from the inaccuracy in determining the position of the DNA ends, and thus the DNA stretch rate. This rate is included in the calculation of the MSD for taking into account the effective translocation of the enzyme along the DNA. Assuming a localization accuracy of ∼120 nm for the end-to-end distance (∼2 µm), we obtained ΔD/D ∼ 0.15, which was translated into error bars on the MSD plots (Figure 3).

Bottom Line: We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6.Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D(1)), revealing a significant size dependency.The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Kastler Brossel, ENS, UPMC-Paris6, CNRS UMR 8552, 24 rue Lhomond, 75005 Paris, France. jasmina.dikic@lkb.ens.fr

ABSTRACT
It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE-DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D(1)), revealing a significant size dependency. To account for the dependence of D(1) on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts.

Show MeSH
Related in: MedlinePlus