The helical domain of the EcoR124I motor subunit participates in ATPase activity and dsDNA translocation

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested

Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling

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

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

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

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