(A) Overall structure of UL89-C in a ribbon representation. The metal ions are indicated by yellow spheres. UL56-interacting helix α4 is highlighted in blue, α helices in cyan, 3₁₀ helices in green, and β strands in magenta. (B) Detailed view of the active site area. One Mn²⁺ ion is shown coordinated by Asp463 and Glu534 and a second one is coordinated by Asp463 and Glu651. (C) Topology diagram of UL89. Color code as in (A).

UL89-C in vitro nuclease assay. (A) Effect of divalent cations on UL89-C linear dsDNA nuclease activity. Lane 1: linear (digested with HindIII) pUC18 plasmid in the absence of the nuclease. Lane 2: nuclease reaction in the absence of divalent cations. Lane 3: nuclease reaction in the presence of Mg²⁺. Lane 4: nuclease reaction in the presence of Ca²⁺. Lane 5: nuclease reaction in the presence of Mn²⁺. (B) Effect of divalent cations on UL89-C circular dsDNA nuclease activity. Lane 1: circular pUC18 plasmid in the absence of the nuclease. Lane 2: linear (digested with HindIII) pUC18 plasmid in the absence of the nuclease. Lane 3: nuclease reaction in the absence of divalent cations. Lane 4: nuclease reaction in the presence of Mg²⁺. Lane 5: nuclease reaction in the presence of Ca²⁺. Lane 6: nuclease reaction in the presence of Mn²⁺. (C) Nuclease assays with the UL89-C WT and mutant proteins with linear dsDNA. Lane 1: linear (digested with HindIII) pUC18 plasmid in absence of the nuclease. Lane 2: nuclease assay with the WT proteins. Lane 3: nuclease assay with the D463A mutant. Lane 4: nuclease assay with the D651A mutant. Lane 5: nuclease assay with the D463A E534A mutant. (D) Nuclease assay with the UL89-C WT and mutant proteins with ds-circular DNA. Lane 1: circular pUC18 plasmid in absence of the nuclease. Lane 2: nuclease assay with the WT protein. Lane 3: nuclease assay with the D463A mutant. Lane 4: nuclease assay with the D651A mutant. Lane 5: nuclease assay with the D463A E534A mutant. Mutant proteins show only residual nuclease function. Labels: sc, supercoiled circular plasmid DNA; ni-oc, nicked open circular plasmid DNA; li, linear plasmid DNA.

UL89-C surface representations. (A) Conservation surface of UL89-C based on a Risler matrix calculation. Residue color is shown on the basis of conservation score within the eight human herpesvirus: orange, more than 80% similarity; green, full conservation. Metal ions are shown in yellow. (B) Electrostatic potential surface of UL89-C in the same view as (A). The positive potential is shown in blue whereas the negative potential is in red.

Inhibition of UL89-C nuclease activity by raltegravir. Lane 1: Circular pUC18 plasmid in the absence of the nuclease. Lane 2: Linear (digested with HindIII) pUC18 plasmid in the absence of the nuclease. Lane 3: Nuclease assay with the UL89-C WT protein. Lanes 4–14: Nuclease assays in the presence of a range of concentrations of raltegravir (0.5, 1, 2, 4, 8, 16, 32, 64, 128, 256 and 512 μM).