Crystallographic data collection and refinement statistics.

<p>* Values in parentheses are for the highest-resolution shell.</p><p>Crystallographic data collection and refinement statistics.</p

Resolved 180s loop.

<p>Electron density (blue mesh) in Lys220Ala mutant HsdR is shown only for the 180s loop, with selected sidechains of the loop shown as sticks in atomic colors with yellow carbons. Outside the 180s loop the sidechains of residues Asp151, Glu165, and Lys167 in the active site, and of Ala220 and Asn221 in the 220s loop, are labeled and shown as sticks, and alpha helix 7 and beta strand f are labeled. The dashed line indicates a distance short enough to permit bonding between the indicated functional groups. Coloring as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128700#pone.0128700.g006" target="_blank">Fig 6</a>.</p

ATP contacts.

<p>Models and electron density are shown for A, WT HsdR; B, Lys220Glu chain A; C, Lys220Arg; D, Lys220Ala. Domain segments (ribbons) and selected residues (stick models) are color-coded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128700#pone.0128700.g001" target="_blank">Fig 1</a>, with Mg ion shown as a green sphere. Electron density (blue mesh) is shown for ATP (upper center of each panel, atomic colors and orange carbon) and for the 220s loop (lower). The electron density for WT HsdR is better at the same contour level due to its higher resolution, with corresponding differences in the electron density mesh spacing. Dashed lines indicate distances short enough to permit bonding interactions between the indicated functional groups.</p

DNA binding.

<p>A. Sequence of synthetic 30-basepair oligonucleotide used, with the EcoR124I recognition sequence shown in bold. B. Electrophoretic mobility-shift assay. The oligonucleotide was 5’ end-labeled with polynucleotide kinase and titrated with EcoR124I reconstituted from HsdS₁HsdM₂ methylase and WT or Lys220Glu HsdR. The oligonucleotide concentration is 5 nM, the concentration of methylase (M2S1 complex) is 40nM, and the concentrations of HsdR are 20, 40, 80, and 120 nM, respectively, in lanes 2–5 (WT) and 8–11 (Lys220Glu). Lane 6, DNA only; lanes 1 and 7, DNA and methylase only. The numbers of subunits in each DNA-protein complex are indicated on the right: R, motor subunit HsdR; M, methylase subunit HsdM; S, specificity subunit HsdS.</p

Effect of changes at HsdR Lys220 on the restriction phenotype of EcoR124I.

<p>^a Restriction activity was determined as the efficiency of plating of λvir.0 on the test strains relative to the efficiency of plating of λvir.0 on <i>E</i>. <i>coli</i> JM109(DE3) indicator (nonrestricting) strain as described in Methods. The values are the mean of at least three independent experiments. ^SD The standard deviation</p><p>^b Positive complementation was tested in r− host <i>E</i>. <i>coli</i> JM109(DE3)[pACMS] (r−m+).</p><p>^c Negative complementation was tested in r+ host <i>E</i>. <i>coli</i> JM109(DE3)[pKF650] (r+m+).</p><p>Effect of changes at HsdR Lys220 on the restriction phenotype of EcoR124I.</p

Cleavage of circular DNA.

<p>Circular plasmid DNA bearing one EcoR124I recognition site was reacted with enzymes reconstituted from HsdS₁HsdM₂ methylase and WT HsdR or mutant HsdRs Lys220Glu, Lys220Ala, or Lys220Arg, and analyzed as in Fig 2. OC, open circular product (▲); L, linear product (●); SC, supercoiled substrate (■); C, control plasmid linearized with HindIII. A: Reactions stopped at the indicated time points were applied to 1.2% agarose gels and visualized by ethidium bromide staining. M is the marker of the indicated numbers of basepairs and C is the linearized plasmind DNA as a control B: Quantification. The three indicated DNA species were quantified individually. Plots for the increase of linear DNA product were derived by fitting an exponential rise to maximum function in SigmaPlot. The points are given for quantification of the gels shown in A, and standard deviations are given from the mean of seven repetitions (WT, Lys220Ala, Lys220Glu) or six repetitions (Lys220Arg) of the experiment conducted with independently purified enzyme preparations.</p

DNA-dependent ATPase activity.

<p>EcoR124I reconstituted from methylase and WT (blue) or mutant HsdRs Lys220Ala (green), Lys220Glu (red), or Lys220Arg (black) was incubated at a final concentration of 15 nM with 90 nM circular plasmid DNA containing one recognition site and 2 mM ATP containing 0.16 μCi g-³²P-ATP. At the indicated time points ATP and inorganic phosphate were resolved on cellulose TLC, autoradiographed, and scanned to quantify the extent of hydrolysis. For clarity error bars are shown for WT only as they would overlay, nevertheless they are of similar dimension for all mutants.</p