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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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(A) A schematic display of different labels attached to EcoRV. The 2D representation of EcoRV and DNA is drawn around the crystal structure of the EcoRV/DNA complex (protein data bank 4rve). The labels are attached to the enzyme via flexible polyethylene glycol linkers (PEG2 or PEG11) at residue 58, or via the N-terminal polyhistidine His6-tag (located in the vicinity of the position 58). Radii of different labels are: Cy3B ∼ 0.5 nm, sav-Cy3 ∼ 2.1 nm, QDEO6 ∼ 7.2 nm, QD605 ∼ 10 nm, QD655 ∼ 15.4 nm. EcoRV and labels are drawn to scale. (B) The longitudinal MSD of EcoRV labeled with QDE06 or QD605 via PEG linkers of different length. The MSD depends linearly on time, which shows that the QD labeled enzyme slides along the DNA. The linear diffusion constant D1 is derived from the slope of the curve (dashed lines: linear fits on the first five points of the MSD) using the relation: slope = 2D.
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gkr1309-F1: (A) A schematic display of different labels attached to EcoRV. The 2D representation of EcoRV and DNA is drawn around the crystal structure of the EcoRV/DNA complex (protein data bank 4rve). The labels are attached to the enzyme via flexible polyethylene glycol linkers (PEG2 or PEG11) at residue 58, or via the N-terminal polyhistidine His6-tag (located in the vicinity of the position 58). Radii of different labels are: Cy3B ∼ 0.5 nm, sav-Cy3 ∼ 2.1 nm, QDEO6 ∼ 7.2 nm, QD605 ∼ 10 nm, QD655 ∼ 15.4 nm. EcoRV and labels are drawn to scale. (B) The longitudinal MSD of EcoRV labeled with QDE06 or QD605 via PEG linkers of different length. The MSD depends linearly on time, which shows that the QD labeled enzyme slides along the DNA. The linear diffusion constant D1 is derived from the slope of the curve (dashed lines: linear fits on the first five points of the MSD) using the relation: slope = 2D.

Mentions: First, we conjugated fluorescent labels varying in size by a factor of 30 (Figure 1A) to the K58C variant of EcoRV. We estimated the hydrodynamic radius (rl) of the different labels using fluorescence correlation spectroscopy (FCS) (Supplementary Figure S1, Supplementary Table S1). The organic dye Cy3B (rl ∼ 0.5 nm) was attached to EcoRV via a single cysteine residue. The fluorescent protein streptavidin-Cy3 (savCy3, rl ∼ 2.1 nm), and commercial streptavidin/polymer coated QDs (QD605, rl ∼ 10.0 nm; QD655, rl ∼ 15.4 nm) were attached to EcoRV by biotinylating EcoRV at the same cysteine residue (C58). Smaller QDs (QDEO6, rl ∼ 7.2 nm) than those available commercially were prepared with a coating of short hydrophilic peptides, and EcoRV was directly conjugated through its N-terminal His6-tag to the nanoparticle surface (24).Figure 1.


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) A schematic display of different labels attached to EcoRV. The 2D representation of EcoRV and DNA is drawn around the crystal structure of the EcoRV/DNA complex (protein data bank 4rve). The labels are attached to the enzyme via flexible polyethylene glycol linkers (PEG2 or PEG11) at residue 58, or via the N-terminal polyhistidine His6-tag (located in the vicinity of the position 58). Radii of different labels are: Cy3B ∼ 0.5 nm, sav-Cy3 ∼ 2.1 nm, QDEO6 ∼ 7.2 nm, QD605 ∼ 10 nm, QD655 ∼ 15.4 nm. EcoRV and labels are drawn to scale. (B) The longitudinal MSD of EcoRV labeled with QDE06 or QD605 via PEG linkers of different length. The MSD depends linearly on time, which shows that the QD labeled enzyme slides along the DNA. The linear diffusion constant D1 is derived from the slope of the curve (dashed lines: linear fits 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-F1: (A) A schematic display of different labels attached to EcoRV. The 2D representation of EcoRV and DNA is drawn around the crystal structure of the EcoRV/DNA complex (protein data bank 4rve). The labels are attached to the enzyme via flexible polyethylene glycol linkers (PEG2 or PEG11) at residue 58, or via the N-terminal polyhistidine His6-tag (located in the vicinity of the position 58). Radii of different labels are: Cy3B ∼ 0.5 nm, sav-Cy3 ∼ 2.1 nm, QDEO6 ∼ 7.2 nm, QD605 ∼ 10 nm, QD655 ∼ 15.4 nm. EcoRV and labels are drawn to scale. (B) The longitudinal MSD of EcoRV labeled with QDE06 or QD605 via PEG linkers of different length. The MSD depends linearly on time, which shows that the QD labeled enzyme slides along the DNA. The linear diffusion constant D1 is derived from the slope of the curve (dashed lines: linear fits on the first five points of the MSD) using the relation: slope = 2D.
Mentions: First, we conjugated fluorescent labels varying in size by a factor of 30 (Figure 1A) to the K58C variant of EcoRV. We estimated the hydrodynamic radius (rl) of the different labels using fluorescence correlation spectroscopy (FCS) (Supplementary Figure S1, Supplementary Table S1). The organic dye Cy3B (rl ∼ 0.5 nm) was attached to EcoRV via a single cysteine residue. The fluorescent protein streptavidin-Cy3 (savCy3, rl ∼ 2.1 nm), and commercial streptavidin/polymer coated QDs (QD605, rl ∼ 10.0 nm; QD655, rl ∼ 15.4 nm) were attached to EcoRV by biotinylating EcoRV at the same cysteine residue (C58). Smaller QDs (QDEO6, rl ∼ 7.2 nm) than those available commercially were prepared with a coating of short hydrophilic peptides, and EcoRV was directly conjugated through its N-terminal His6-tag to the nanoparticle surface (24).Figure 1.

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