Figure 4. cAAA-stimulated NucC hexamerization is required for DNA cleavage activity and bacteriophage λ immunity.

(A) Cartoon view of two cAAA-bound NucC trimers (colored violet/gray/white and light blue, respectively) assembled into a homohexamer. Bound cAAA molecules are shown in yellow sticks. (B) Closeup view of the NucC α0-α0 interface mediating hexamer formation. (C) Closeup view of the NucC hydrophobic cluster interface mediating hexamer formation. (D) Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) for Ec NucC in the absence of cAAA (gray line with molecular weight measurement in cyan) and in the presence of cAAA (black line with molecular weight measurement in violet). The molar mass of an Ec NucC homotrimer is 80.1 kDa, and a homohexamer is 160.3 kDa. See Figure S5G for SEC-MALS of Pa NucC in the presence and absence of cAAA. (E) SEC-MALS analysis of Ec NucC L19D mutant, which remains trimeric in the presence of cAAA. See Figure S6A–F for SEC-MALS analysis of other Ec NucC mutants. (F) Plasmid digestion assay with wild-type and F28A mutant Ec NucC (10 nM), in the presence of cAAA (for each set: 400, 100, 25, 6.25, and 0 nM cAAA). “L” denotes linear plasmid, “SC” denotes closed-circular supercoiled plasmid, and “cut” denotes fully-digested DNA. See Figure S6G for plasmid digestion data on other Ec NucC mutants. (G) Dilutions of bacteriophage λ on lawns of E. coli MS115–1 (top) and JP313 (wild-type laboratory strain) with the plasmid-encoded E. coli MS115–1 CBASS system encoding wild-type proteins or the indicated mutations. EV: empty vector. Six 10-fold bacteriophage dilutions are shown. (H) Summary of SEC-MALS, in vitro DNA cleavage activity, and bacteriophage λ immunity data for Ec NucC hexamer interface mutants. Shaded in gray are mutants that strongly disrupt both hexamer formation and DNA cleavage; NucC F28A further disrupts bacteriophage λ immunity. N/D: not determined.