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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.

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Related in: MedlinePlus

(A) One possible model of EcoRV (protein data bank 4rve) fused to scRM6 protein (protein data bank 1qx8), in which the structure of scRM6 protein is aligned with the N-terminal helix of EcoRV. EcoRV is presented in magenta, DNA in green, scRM6 protein in gray and the label in orange. (B) The longitudinal (along the DNA molecule) and transverse (perpendicular to the DNA molecule) MSD of EcoRV fused to the scRM6 protein labeled with Cy3B. The longitudinal MSD depends linearly on time, which shows that the fusion protein slides along the DNA, while, as expected, the transverse MSD is constant over time. The linear diffusion constant D is derived from the slope of the curve (dashed line: linear fit on the first five points of the MSD) using the relation: slope = 2D.
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gkr1309-F2: (A) One possible model of EcoRV (protein data bank 4rve) fused to scRM6 protein (protein data bank 1qx8), in which the structure of scRM6 protein is aligned with the N-terminal helix of EcoRV. EcoRV is presented in magenta, DNA in green, scRM6 protein in gray and the label in orange. (B) The longitudinal (along the DNA molecule) and transverse (perpendicular to the DNA molecule) MSD of EcoRV fused to the scRM6 protein labeled with Cy3B. The longitudinal MSD depends linearly on time, which shows that the fusion protein slides along the DNA, while, as expected, the transverse MSD is constant over time. The linear diffusion constant D is derived from the slope of the curve (dashed line: linear fit on the first five points of the MSD) using the relation: slope = 2D.

Mentions: Next, we fused two engineered Rop proteins (scRM6) to one EcoRV protein, each at the N-terminal α-helix of the two EcoRV subunits, and further labeled this fusion construct with Cy3B (Figure 2A). From the hydrodynamic radius of the fusion construct, measured using FCS (Supplementary Figure S2), we derived an effective radius rl ∼ 3.1 nm for each of the scRM6 proteins.Figure 2.


The rotation-coupled sliding of EcoRV.

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

(A) One possible model of EcoRV (protein data bank 4rve) fused to scRM6 protein (protein data bank 1qx8), in which the structure of scRM6 protein is aligned with the N-terminal helix of EcoRV. EcoRV is presented in magenta, DNA in green, scRM6 protein in gray and the label in orange. (B) The longitudinal (along the DNA molecule) and transverse (perpendicular to the DNA molecule) MSD of EcoRV fused to the scRM6 protein labeled with Cy3B. The longitudinal MSD depends linearly on time, which shows that the fusion protein slides along the DNA, while, as expected, the transverse MSD is constant over time. The linear diffusion constant D is derived from the slope of the curve (dashed line: linear fit on the first five points of the MSD) using the relation: slope = 2D.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr1309-F2: (A) One possible model of EcoRV (protein data bank 4rve) fused to scRM6 protein (protein data bank 1qx8), in which the structure of scRM6 protein is aligned with the N-terminal helix of EcoRV. EcoRV is presented in magenta, DNA in green, scRM6 protein in gray and the label in orange. (B) The longitudinal (along the DNA molecule) and transverse (perpendicular to the DNA molecule) MSD of EcoRV fused to the scRM6 protein labeled with Cy3B. The longitudinal MSD depends linearly on time, which shows that the fusion protein slides along the DNA, while, as expected, the transverse MSD is constant over time. The linear diffusion constant D is derived from the slope of the curve (dashed line: linear fit on the first five points of the MSD) using the relation: slope = 2D.
Mentions: Next, we fused two engineered Rop proteins (scRM6) to one EcoRV protein, each at the N-terminal α-helix of the two EcoRV subunits, and further labeled this fusion construct with Cy3B (Figure 2A). From the hydrodynamic radius of the fusion construct, measured using FCS (Supplementary Figure S2), we derived an effective radius rl ∼ 3.1 nm for each of the scRM6 proteins.Figure 2.

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