Extended Data Fig. 5: DdmD is helicase-nuclease.
From: Plasmid targeting and destruction by the DdmDE bacterial defence system
a & b, TBE-Urea PAGE gel analysis of DNA cleavage by DdmD in the presence and absence of ATP. A 3′-FAM-labeled DNA substrate was incubated with DdmD as ssDNA (left), annealed to a partially complementary strand (creating a forked duplex with a 30-nt overhang and a 25-bp duplex), and a 5′ overhang substrate (through annealing to a 25-nt complementary strand). Within the structures of DdmD bound to DNA, the 3′ end occupies the RecA helicase channel. ssDNA and forked DNA substrates are degraded, while a larger, incomplete degradation product for the 5′ overhang substrate is observed, since only the ssDNA overhang itself can be cleaved by DdmD. This indicates that unwinding and translocation is essential for full duplex degradation by DdmD. Representative of three independent experiments. b, Nuclease assay as in a, but the FAM is on the 5′ single-stranded end of the forked substrate. Since ssDNA is readily cleaved by DdmD, complete degradation is observed in the absence of ATP. Representative of three independent experiments. c, DdmD DNA unwinding assay, where a fluorophore-quencher pair (FAM and BlackHole Quencher) are on each strand of the forked substrate. DdmD unwinding is ATP-dependent. Data are mean ± s.d. of at three technical replicates d, Gel-based unwinding assay. ATP and DdmD are required for duplex unwinding, as monitored using native TBE 10% PAGE gel. e, Visualization of DNA-bound dimeric (left) and monomeric (right) complex by cryoEM. The monomeric DNA-bound DdmD suffers from severe preferred orientation. Representative of three independent experiments. f, Native PAGE gel analysis of DdmD oligomeric state in the absence and presence of DNA substrates. Apo DdmD runs as a dimeric species, which shifts to a mixture of monomer and dimer with ssDNA, and predominantly monomer with the 30-nt overhang forked DNA substrate used for structural analysis in Fig. 2. Representative of three independent experiments. g, Negative stain EM 2D classes of DdmD in complex with DNA substates used for panel f. h, Visualization of DNA-bound dimeric (left) and monomeric (right) complex by cryoEM. The monomeric DNA-bound DdmD suffers from severe preferred orientation. i, SEC chromatogram of DdmDE handover complex (as shown in Fig. 3a, green), and handover complex reconstituted with DdmD(R620A) point mutant (pink). A280nm absorbance has been normalized to the size of the largest peak. The DdmD(R620A)E HC peak (peak i) is much smaller than wild-type, and unbound, dissociated DdmE is present (peak ii). Free DNA is in peak iii*, and has an A260nm/A280nm ratio of 1.8, while peaks i and ii had ratios of 1.2 and 1.1, respectively. SDS-PAGE analysis of DdmD(R620A)E HC SEC fractions is shown below. For gel source data, see Supplementary Fig. 1.
