- Measurement of the contributions of 1D and 3D pathways to the translocation of a protein along DNA

mcith_zpq90001.jpg Figure 1 Experimental strategy. Shown is a DNA with two BbvCI sites 30 bp apart, in either directly repeated (a) or inverted (b) orientations, as marked by the arrowheads. The points of cleavage in each site are marked by white arrows connected by a jagged line. The distances between the scissile bonds are indicated for both CC → CC and GC → GC separations. In both a and b, the BbvCI restriction endonuclease is shown bound to both sites: its R1 and R2 subunits are shown as blue and green triangles, respectively, with dark blue and dark green hemispheres for their catalytic centers. The subunits are placed on their target strands, R1 on GC and R2 on CC. In both a and b, the minimal motion needed to reposition the enzyme from the left-hand to the right-hand site is shown with red arrow(s): a linear translocation between the directly repeated sites in a and a rotation around and a turnover along the DNA with the inverted sites in b.

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mcith_zpq90002.jpg Figure 2 BbvCI reactions. (a) Shown as ABC is a 301-bp DNA with two BbvCI sites in inverted orientation 75 bp apart. The sites, 1 and 2, have the scissile bonds in the CC sequence at positions 101 and 173, respectively. The initial reaction of BbvCI on this DNA has three possible outcomes: cleavage at site 1 alone to give fragments A (in black) and BC (in blue); at 2 alone for fragments AB (in red) and C (in green); processive cutting at both 1 and 2 to give fragments A, B (in purple), and C. (b and c) BbvCI endonuclease (0.3 nM) was added to the above DNA (32P-labeled, 30 nM) in Reaction Buffer at 37°C. Samples were taken at the indicated times and subjected to electrophoresis through polyacrylamide. b shows a phosphorimager record of the gel, annotated on the right with the mobilities (and sizes) of all of the DNA species from the reaction. For c, the concentrations of the following fragments were evaluated from the record: A, black diamonds; BC, blue circles; AB, red triangles; C, green squares. The dotted lines through each data set are the optimal fits to linear slopes to give, respectively, vA, vBC, vAB, and vC.

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mcith_zpq90003.jpg Figure 3 Processivity ratios. The reactions contained BbvCI and a DNA with two sites in repeated or inverted orientations separated by one of the distances noted below and NaCl as indicated. For each intersite distance and NaCl concentration, the R/I ratio of fP values on repeated sites to inverted sites was calculated as follows: sites 30 bp apart, black circles and line; 40 bp, white triangles; 45 bp, white squares; 75 bp, black diamonds and line.

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