CasDinG is a 5′-3′ dsDNA and RNA/DNA helicase with three accessory domains essential for type IV CRISPR immunity

Abstract CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Here, we demonstrate that CasDinG from Pseudomonas aeruginosa strain 83 is an ATP-dependent 5′-3′ DNA translocase that unwinds double-stranded (ds)DNA and RNA/DNA hybrids. The crystal structure of CasDinG reveals a superfamily 2 helicase core of two RecA-like domains with three accessory domains (N-terminal, arch, and vestigial FeS). To examine the in vivo function of these domains, we identified the preferred PAM sequence for the type IV-A system (5′-GNAWN-3′ on the 5′-side of the target) with a plasmid library and performed plasmid clearance assays with domain deletion mutants. Plasmid clearance assays demonstrated that all three domains are essential for type IV-A immunity. Protein expression and biochemical assays suggested the vFeS domain is needed for protein stability and the arch for helicase activity. However, deletion of the N-terminal domain did not impair ATPase, ssDNA binding, or helicase activities, indicating a role distinct from canonical helicase activities that structure prediction tools suggest involves interaction with dsDNA. This work demonstrates CasDinG helicase activity is essential for type IV-A CRISPR immunity as well as the yet undetermined activity of the CasDinG N-terminal domain.


INTRODUCTION
CRISPR-Cas systems are prokaryotic immune systems that protect against mobile genetic elements, such as viruses and plasmids ( 1 , 2 ). Of the six known CRISPR system types, type IV systems are the least understood and display a large di v ersity of protein composition (3)(4)(5). Type IV-A systems rely on a multi-subunit complex consisting of a CRISPRderi v ed RN A (crRN A) bound by se v eral Type IV-specific CRISPR-associated proteins (Csf1, Csf2, Csf3, Csf5 / Cas6), and the CRISPR-associated DinG (CasDinG) protein to clear invasi v e plasmids (6)(7)(8) (Figure 1 A). Mutating either the CasDinG Walker A or Walker B motifs, predicted to bind and hydrolyze ATP, impaired type IV immunity ( 7 , 8 ), suggesting CasDinG-mediated ATP binding and hydrolysis are essential for type IV-A function.
DinG ( damag e-inducib le g ene G ) proteins are superfamily 2 (SF2) helicases related to eukaryotic XPD helicases involved in nucleotide excision repair (9)(10)(11). Deletion of the dinG gene in E. coli results in ultraviolet radiation sensitivity and mutations in the human XPD protein cause heritab le ultrasensiti vity to ultraviolet light and pr ematur e cancers ( 12 , 13 ). Notably, in bacteria DinG orthologs display variability in domain organization and the functional role of ATP binding and hydrolysis. For example, E. coli DinG is a 5 -3 DNA helicase that relies on ATP hydrolysis and an accessory domain containing an iron-sulfur (FeS) cluster to unwind substrates (13)(14)(15), while S. aureus DinG lacks an FeS cluster domain and does not unwind DNA ( 16 ). Instead, S. aureus DinG contains an N-terminal accessory domain with 3 -5 exonuclease activity that is regulated by ATP binding.
CasDinG proteins are distinct from non-Cas DinG sequences ( 17 ), including the E. coli and S. aureus DinG proteins described above (Supplementary Figure S1). For example, CasDinG contains an accessory domain in the same location as the E. coli FeS cluster domain, but the amino acid sequence of the CasDinG domain lacks cysteines for coordinating an FeS cluster ( 17 ). Additionally, CasDinG has an N-terminal accessory domain, but it is smaller than the S. aureus N-terminal exonuclease domain and bioinformatic studies do not predict the domain to harbor an exonuclease activity (Supplementary Figure S1) ( 4 , 5 ). Thus, sequence comparisons to previously investigated DinG proteins could not fully re v eal the biochemical function of CasDinG.
Nucleic Acids Research, 2023, Vol. 51, No. 15 8117 To better understand the function of CasDinG in type IV-A CRISPR immunity, we performed ATPase, nucleic acid binding, and helicase assays with recombinant CasD-inG protein encoded on a P. aeruginosa strain 83 extrachromosomal plasmid (NCBI r ef: NZ CP017294.1), r eferr ed to throughout the paper as CasDinG (Figure 1 A). We also solved the x-ray crystal structure of CasDinG, defined the type IV-A consensus protospacer adjacent motif (PAM) with a plasmid curing library, and performed cell-based assays with single point mutations and domain deletion mutants. Our results indicate CasDinG is an ATP-dependent 5 -3 DN A translocase ca pable of unwinding DN A and RN A / DN A duplexes comprised of an SF2 helicase core and three accessory domains. Plasmid curing assays indicate each of these domains (N-terminal, vFeS, and arch) is essential for type IV-A imm unity. Notabl y, the removal of the N-terminal domain inhibited type IV-A immunity, but did not disrupt ATPase or helicase activities, indicating the N-terminal domain serves an essential function in type IV-A immunity beyond substrate unwinding. Predicted structures of the N-terminal domain suggest it adopts a fold observed in dsDNA binding proteins, indicating the Nterminal domain may interact directly with dsDNA. This w ork pro vides a structural and biochemical f oundation f or understanding the role of CasDinG in type IV-A CRISPR immunity and re v eals that the N-terminal domain of Cas-DinG plays an essential but unclear role that may hold the key to understanding the elusi v e type IV-A immune systems.

Construct gener ation, expr ession & purification of full length and domain deletion proteins
The dinG gene from a P. aeruginosa strain 83 extrachromosomal plasmid (NCBI ref: WP 088922490.1 (protein sequence), NZ CP017294.1 (plasmid sequence)) was synthesized by TWIST bioscience and cloned into a pET StrepII TEV ligation independent cloning (LIC) vector (2R-T) ( 7 ). The annotated gene begins with a non-canonical (TTG) start codon, but the open reading frame continues se v eral amino acids upstream of the gene annotation. Thus, to ensure we were expressing the entire biolo gicall y relevant sequence we included the in-frame sequence upstream of the annotation starting with another non-canonical (TTG) start codon befor e r eaching a stop codon. This approach added 48 DNA bases to the annotated gene and 16 additional amino acids (MKLAQGAFVDVIRIGA) to the N-terminus of the annotated amino acid sequence. Additionally, with this modification, the currently annotated TT G start codon w ould encode for a leucine instead of methionine.
Domain deletion constructs for recombinant expression and cell-based assays were generated using the NEBaseChanger tool to design primers and the NEB Q5 Site-directed Mutagenesis Kit (E0554). For the N-terminal domain deletion, 306 bases (encoding amino acids 2-103) wer e r emoved. For the vFeS domain deletion, bases encoding amino acids 200-263 wer e r eplaced with bases encoding three glycines. For the vFeS loop deletion, the bases encod-ing amino acids 227-246 were replaced with bases encoding tryptophan, aspartate, and glycine (the amino acid sequence observed in EcDinG in this region). For the arch domain deletion, bases encoding residues 351-464 were removed, directly linking amino acids 350-465.
Vectors were transformed into E. coli BL21 HMS174(DE3) chemically competent cells (Novagen). A colony was picked and placed into an overnight outgrowth in Luria-Bertani (LB) media a t 37 • C . In a 2.8 l flask, 1 l of LB medium supplemented with 1 ml of 1000 × metals mix (0.1 M FeCl 3 -6H 2 (1 M) was inoculated with 20 ml of overnight starter. Cells were grown to an optical density between 1.0 and 1.3 OD 600 a t 37 • C , then induced with a final concentration of 0.5 mM IPTG (isopropyl ␤-D -1-thiogalactopyranoside), while dropping the temperature to 20 • C. After 5 h, cells were harvested via high-speed centrifugation and stored at -80 • C.
Cells were homogenized on ice with lysis buffer (100 mM Tris Base pH 8.0, 150 mM NaCl, 1 mM TCEP). Protease inhibitors Aprotinin 1000 × (0.5 mg / ml), Leupeptin 1000 × (0.5 mg / ml), Pepstatin A 1000 × (0.7 mg / ml), & PMSF (phen ylmethylsulf on yl fluoride) 150 × (25 mg / ml) were added before cell lysis. Probe sonication for cell lysis was performed at settings of 4 / 60 (power / output). The lysate was clarified by high-speed centrifuga tion a t 16 000 RPM for 30 min. All purification steps were performed a t 4 • C . If the construct contained a Strep-II tag the supernatant was loaded onto strep resin (Strep-Tactin ®XT 4Flow ®, IBA). The resin w as w ashed with lysis buffer and then eluted with Strep elution buffer (100 mM Tris Base, 150 mM NaCl, 50 mM biotin, 1 mM TCEP, pH 8.0). If the construct contained a His-Sumo tag or His tag, the supernatant was loaded onto Ni-NTA resin and was eluted with His elution buffer (100 mM Tris Base, 150 mM NaCl, 500 mM imidazole, 1 mM TCEP, pH 8.0). Fractions with CasDinG were pooled and desalted (HiPrep 26 / 10 Desalting, GE Healthcare) into the low salt buffer (100 mM Tris Base pH 8.0, 10 mM NaCl, 1 mM TCEP). Elutions were then run over a heparin column (HiTrap Heparin HP, GE Healthcare), washed with 47.5 mM NaCl buffer, and eluted with a high salt buffer (100 mM Tris Base pH 8.0, 500 mM NaCl, 1 mM TCEP). When present, the His-Sumo tag was removed by incubating with Tabacco Etch Virus (TEV) protease at a 50:1 (CasDinG: TEV protease Absorbance 280 ra tio) a t 4 • C and then putting the cleaved CasDinG over Ni-NTA r esin to r emov e the cleav ed tag or uncleav ed protein.
Samples were spin concentrated (Corning ® Spin-X ® UF 50 MWCO) before further purification with size exclusion (HiLoad 26 / 600 Super de x 200 pg., GE Healthcare) into the high salt buffer (100 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP). Protein samples were concentrated and stored at 4 • C. Protein was assessed for purification after each step via 12% SDS-PAGE. Notably, we observed that glycerol and / or freezing of the purified protein impaired CasDinG enzymatic activities.
Protein concentration was determined by UV-Vis spectroscopy (Thermofisher UV-vis Nanodrop), using the Beer-Lambert law to correct absorbance values for extinction coefficient as determined by Expasy Protparam ( 18 ).

Clustal omega alignments and EMBOSS NEEDLE pairwise alignments
E. coli (P27296) and S. aureus (Q2FGY5) DinG sequences were obtained through the Uniprot database server and aligned to P. aeruginosa 83 CasDinG using the Clustal Omega multiple sequence alignment tool ( 19 ). Pairwise sequence alignments were performed between Pa83 CasDinG and E. coli or S. aureus sequences using an EMBOSS Needle alignment ( 20 ). Structural alignments between domains were either performed with the Secondary Structure Matching tool in Coot ( 21 , 22 ), the Dali server ( 23 ), or the align command in the PyMOL Molecular Graphics System, Version 2.0 Schr ödinger, LLC.

Nucleic acid substrate preparation
Nucleic acids were synthesized by Integrated DNA Technologies (IDT) (Supplementary Table S1). Nucleic acids were labeled with a fluorescein (FAM) label on the 5 or 3 end by IDT. To make duplexed nucleic acids, complementary oligonucleotides were mixed in an equimolar ratio in the presence of NEB buffer 2.1 and heated to 95 • C. These oligonucleotides were slowly cooled to room temperature before being run on 12-15% NATIVE PAGE gels. Duplex bands were then gel extracted, ethanol precipitated, and reconstituted in water. For a list of oligonucleotides used in assays, refer to Supplementary Table S1.

Malachite green ATPase assays
Concentrations of P i were determined with a Malachite Green Phosphate Assay kit (BioAssay Systems, Hayward, CA, USA). Activated Malachite Green reagent was added to wells of a 384 well plate (Corning Assay Plate, 384 wells, Black with clear bottom, non-binding surface, Low flange, no lid, polystyr ene, 3766). Befor e the r eaction, assay components wer e pr e-incuba ted a t 37 • C for 10-15 min. Reactions were started with the addition of ATP and were run a t 37 • C . The reaction was quenched in the activated Malachite Gr een r eagent at time points, between 30 and 150 s. The quenched reactions were de v eloped for 30 min before sample measurement. The absorbance values of the samples were obtained using a Synergy H4 Hybrid Multi-Mode Microplate Reader measuring absorbance at 620 nm.
Comparison of initial velocities with various nucleic acid substrates utilized 10 nM CasDinG, 100 nM nucleic acid, and 600 M ATP tha t was reconstituted in buf fer (50 mM Tris pH 7. Michaelis-Menten curves were generated for CasDinG (10 nM) with single-stranded DNA (100 nM) and without (basal). Michaelis-Menten kinetics utilized the ATPase buffer (50 mM Tris pH 7.5, 0.1 mg / ml of Recombinant Albumin or Bovine Serum Albumin, 1 mM MgCl 2 , 0.4 mM TCEP). Initial velocities were performed with increasing concentrations of ATP (11-2000 M), and a standard curve was generated using known concentrations of orthophosphate (0-100 M). Initial velocities were calculated using linear r egr ession. For a list of oligonucleotides used in these assays, see Supplementary Table S1. GraphPad Prism for Windows version 9.3.0 was used to fit the data using nonlinear r egr ession to the Michaelis-Menten equation (Equation 1 ). Where is the initial reaction velocity of the reaction, [ S ] is the ATP concentration, K M is the Michaelis constant, and V max is the maximum velocity of the enzyme.
ATP concentrations were spectroscopically verified at 280 nm with crystal cuvettes and a spectrophotometer. Concentrations were confirmed using the Beer-Lambert law (Equa tion 2 ). W here A is the absorption, ε is the extinction coefficient in M −1 cm −1 , l is the path length (cm), and c is concentration (M). The molar extinction coefficient used for ATP was 15 400 M −1 cm −1 . Stock ATP was frozen at -80 • C. Time courses. 15 nM FAM-labeled nucleic acid substrate was exposed to 25 nM CasDinG or CasDinG mutants over 10 min in the presence of 1 mM MgCl 2 and 1 mM ATP in the helicase buffer (50 mM Tris pH 7.5, 0.1 mg / ml recombinant albumin or bovine serum albumin (BSA), 1 mM TCEP) a t 37 • C . Samples were quenched in 2 × STOP Buffer with 475 nM unlabeled quenching oligo, between 0 and 10 min, and run on 15% or 20% TBE Nati v e PAGE gels. The m utant anal ysis utilized the same nucleic acid substrate as WT CasDinG and an equivalent amount of protein.
Gel analysis. All NATIVE PAGE gels for helicase assays were imaged using a BioRad Imaging system and analyzed using BioRad ImageLab softw are. P ercent unwound were quantified using ImageLab software and the reported data is the average of three experiments, error bars when present r epr esent the standard deviation from the mean. Graphs were made in GraphPad Prism for Windows version 9.3.0.

Nucleic acid-binding assays
Nucleic acid-binding activities of strep-tagged WT CasD-inG and CasDinG mutants were monitored using a fluorescence polarization-based assay. Anisotropy data were collected using a BioTek Synergy H4 Hybrid Multi-Mode Microplate Reader with polarizers and bandpass filters. The polarizers and bandpass filters provided 485 ± 20 nm excitation and detection of fluorescence emission at 528 ± 20 nm. Each reaction (80 l) contained a limiting concentration (10 nM) of 3 FAM-labeled ssDNA (40 nt). CasD-inG and 3 -end FAM-labeled nucleic acid were assayed at room temperature with increasing concentrations of CasD-inG (0-2.5 M) in a binding buffer (100 mM Tris pH 8.0, 1 mM TCEP and 5 mM MgCl 2 ) with or without 1 mM AMP-PNP (Sigma Aldrich). Change in anisotrop y r elati v e to FAM-nucleic acid was plotted as a function of CasDinG concentra tion. The apparent dissocia tion constant ( K d ) for the nucleic acid substrate was determined by fitting the raw data to a single site saturation binding model in GraphPad Prism for Windows version 9.3.0.

Crystallization and structure determination
Strep-tagged CasDinG protein was concentrated to 5 mg / ml and crystallized using 225 mM Imidazole pH 8.0, 3.5% PEG 8000 and 4% sucrose mother liquor with hanging-drop vapor diffusion at room temperature. The crystal used for structure determination was retrie v ed from a drop set up with 1 l (5 mg / ml) of protein solution to 3 l mother liquor. The crystal was then soaked in a cryoprotectant solution composed of 30% ethylene glycol and mother liquor, mounted on a loop, and cooled to 100 K. Diffraction data were collected at the SSRL beamline 9-2. The data wer e index ed, integrated, and scaled using HKL3000 to 2.95 Å resolution with the space group P6 5 ( 24 ). Phases were determined by molecular replacement in Phaser ( 25 , 26 ) with an N-terminally truncated Alphafold structure prediction with Colab ( 27 ), pr epar ed with the Process Predicted Model tool in Phenix to adjust the model B-factors ( 28 ). After obtaining an initial solution with Phaser, RESOLVE was used to obtain a density-modified map ( 29 ). Because the unit cell consisted of ∼70% solvent, density modification significantly improved the electron density maps. Model building was performed in Coot ( 22 ), structures were refined using PHENIX, and validation was performed using Molprobity within PHENIX and the PDB deposition servers ( 28 ).

Pr epar ation of electrocompetent cells for PAM assay
50 l of chemically competent HMS174(DE3) cells were first doubly transformed with roughly 20 ng each of plasmids #1284 (pCDF Pa csf1 csf2 cas6) and #1290 (pACYC PaCR83 csf3 CasDinG) containing the PaIV-A CRISPR immune system with CasDinG (targeting). Another vial of HMS174(DE3) cells was also transformed with plasmids #1284 and #1291 (pACYC-csf3-CasDinG) which lacked a CRISPR (control). Cells were then plated with antibiotic selection by Chloramphenicol and Streptomycin and allowed to grow overnight a t 37 • C . The next day 3 wellisolated colonies were picked from both a targeting plate and a control plate and each colony was used to inoculate a 5 ml overnight growth of LB with selection by chloramphenicol and streptomy cin. Ov ernight growths were carried out in a shaking incubator at 37 • C. The f ollowing da y, 2 ml of each overnight growth was used to inoculate 48 ml of fresh LB with selection antibiotics, and the culture was then allowed to grow until reaching an OD of a pproximatel y 0.25. At this point, 0.1mM IPTG was added to induce expression of the CRISPR-Cas system, and the cells w ere allow ed to grow for another 50 min. The cultur es wer e then transferr ed to 50 ml conical tubes and spun down at 4000 × g at 4 • C for 15 min to pellet them. Pellets were then resuspended in 50 ml of ice-cold sterile MilliQ filtered water followed by another round of centrifuga tion a t 4000 × g at 4 • C for 15 min. Pellets were then resuspended in 2 ml of ice-cold sterile 10% glycerol in a new 15 ml conical tube and spun down again at 4000 × g at 4 • C for 10 min. Cells were resuspended one more time in 460 l of ice-cold 10% glycerol and used immediately for electroporation.

In vivo PAM assay
A lyophilized PAM library in the form of a pET backbone containing a CA01 target sequence flanked on the 5 end by a library of all potential base combinations of the fiv e positions immediately upstream of the target was provided by the Chase Biesel labora tory a t the Helmholtz Center for Infection Research, and resuspended in nuclease-free water to a concentration of roughly 500 ng / l. 1.5 l of the PAM library was then added to 100 l of a vial of fresh electrocompetent cells containing either a functional (targeting) or non-functional (control) immune system. This was done in biological triplicate with three targeting and three control transformations. The cells were then transferred to a prechilled 2 mm electroporation cuv ette. Recov ery media consisting of SOB with 0.1 mM IPTG was pr epar ed for each tr ansformation. Electropor ation was carried out for each cuvette with settings of 2500 V, 25 F, and 400 , with a typical time constant of 4-5 ms. Immediately after a ppl ying the voltage 1 ml of recovery media was added, and the cells wer e transferr ed to 1.5 ml microcentrifuge tubes to recover in the incubator at 37 • C with shaking.
After recovery, 1 ml of each transformation was used to inoculate 50 ml of LB with triple antibiotic selection (chloramphenicol 25 g / ml, streptomycin, and kanamycin 50 g / ml). Liquid cultures were then allowed to grow overnight for 20 h at 37 • C with shaking, and then harvested by centrifugation at 4000 × g at 4 • C for 15 min. Plasmid DNA was then extracted from each pellet with an Omega Bio Tek E.Z.N.A. ® Plasmid DNA Midi kit.

Next-generation sequencing
Primers flanking the insertion site in the plasmid were used to amplify the PAM sequences from the PAM library from each of the three targets and three control samples. To these primers were added the Illumina Truseq sequencing primer sequences. This reaction was perf ormed f or 22 cycles using Q5 (New England Biolabs). These Illumina sequences were then used as the template for a second round of PCR to add indexes and the p5 and p7 Illumina adapter sequences to make finished sequencing libraries. This PCR was perf ormed f or 10 cycles using Q5. These libraries were then sequenced on an Illumina MiSeq sequencer using the 300-cycle v2 chemistry. After dem ultiplexing a pproximately 500 000-600 000 r eads wer e obtained for each sample.

PAM sequence analysis
A short Python script was used to parse the fiv e nucleotides preceding a correct target sequence from each read, and the number of occurrences of each potential NNNNN PAM was counted and exported as a CSV file. Counts were summed for all targeting and control runs, and a depletion score was calculated for each PAM sequence which relates the proportion of all counts of a PAM in the targeting runs to the control runs.

DepletionScore = t ot al cont r ol r eads t ot al t arget ing reads * co ntr o l P AM reads t arget ing P AM reads
All PAM sequences along with their depletion scores were then input into a Krona plot generator ( 30 , 31 ) which was in turn used to generate a PAM wheel.
Colonies from the overnight plates were used to inoculate 25 ml of the pr epar ed LB broth in a 50 ml Falcon tube. These cells were then incuba ted a t 37 • C until an OD 600 between 0.2-0.3 when they were induced with 0.1 mM IPTG (100 M IPTG). Cells were allowed to grow for an additional 45 min at 37 • C before being cold-shocked on ice for 20 min. Cells were then spun down at 2700 × g for 15 min a t 4 • C . The superna tant was decanted, and the cells were resuspended in 12 ml RF1 Buffer (100 mM RbCl, 50 mM MnCl 2 ·4H 2 O, 30 mM Potassium acetate, 15% m / v glycerol). Cells were then allowed to rest on ice for 15 min before being spun down at 870 x G for 15 min. The supernatant was decanted and cells were resuspended in 1 ml of RF2 Buffer (10 mM MOPS, 10 mM RbCl, 75 mM CaCl 2 · 2H 2 O 15% m / v glycerol). The cell solution was incubated on ice for 15 min prior to aliquoting cells in 100 ul volumes and flash freezing at −80 • C.

In vivo plasmid competition assay
Chemically competent HMS174(DE3) cells containing plasmids # 1284 and 1290 were transformed with 30 ng of target or non-target plasmid, #2380 (pET27b CA01 GGAAA) and #1095 (pET27b(+)-nontarget TTT C) r especti v ely. Cells were then heat shocked at 42 • C for 30-40 s, followed by cold shock on ice for 1-3 min. 400 l of LB containing 0.1 mM IPTG were then added to the cold-shocked cells. Cells were then incubated at 37 • C for 45-50 min in a shaking incubator, 200 RPM, followed by plating of cells onto a triple antibiotic LB agar selection plate (Chloramphenicol 25 g / ml, streptomycin and kanamycin 50 g / ml, and IPTG 0.1 mM IPTG). The cells were then spread around by shaking with sterile glass beads per plate and subsequently allowed to dry before being placed in a 37 • C incubator for 24 h. Colonies were then counted manually for analysis.

Circular dichroism
CD spectra were collected for WT strep-tagged DinG and CasDinG mutants at an approximate concentration of 0.5 mg / ml. Protein solutions were diluted into the low salt buffer (100 mM Tris Base pH 8.0, 10 mM NaCl, 1 mM TCEP) prior to data collection. Data were collected using a JASCO model J-1500 spectropolarimeter. CD spectra were collected from 190 to 260 nm at 10 • C using 0.1 cm quartz cuvettes, 1 nm data sampling, a 50 nm / min scan rate, and a 2-second da ta integra tion time. Measurements were converted from machine units to ε units by using the equation ε = × (0.1 × MRW) / ( P × Conc) × 3298, where MRW is mean residue weight, P is pathlength, and Conc is protein concentration, as suggested by Dichrow e b ( 32 ).

Structur e pr ediction and comparison
Alphafold2 Colab was used to predict the atomic structure of the N-terminal domain of CasDinG ( 27 ). The Dali server and Foldseek were used to identify structurally homologous structures in the PDB (Dali) and Alphafold (Foldseek) databases ( 23 , 33 ). PyMoL was used to generate comparati v e figures.

CasDinG is a DNA-dependent ATPase
It was recently demonstrated that mutation of the CasD-inG Walk er A (EAAT GT GKG amino acids 138-146) or Walker B (VDEAHLL amino acids 336-342) motif disrupts type IV-A immunity ( 7 , 8 ). Although the canonical function of these motifs is ATP binding and hydrolysis, it remained unclear if CasDinG uses ATP to unwind DNA duplexes, like E. coli DinG ( 13 ), or to regulate an accessory acti vity, like S . aureus DinG ( 16 ). Thus, to determine the role of ATP in CasDinG function we expressed and purified recombinant CasDinG from P. aeruginosa strain 83 extrachromosomal plasmid (Supplementary Figure S2) , and used a malachite green assay to detect the release of inorganic phosphate from ATP (Figure 1 B). Because ATPase activities of helicases are often enhanced by the presence of nucleic acid ( 34 , 35 ), we examined ATP hydrolysis in the presence of various DNA substrates (Figure 1 B and Supplementary Table S1). We observed that a 17 nt DNA duplex with a 16 nt poly-T 5 -overhang strongly enhanced ATPase activity, while a similar DNA duplex with a 3overhang only moderately enhanced ATPase activity, and a 17 nt blunt end duplex did not enhance ATPase activity above background. These data suggested that CasD-inG ATPase activity is enhanced by nucleic acid, with a pr efer ence for substrates with 5 single-stranded (ss)DNA overhangs.
To determine how nucleic acid length influences CasD-inG ATPase activity, we examined activity in the presence of a 17 and 40 nt random sequence ssDNA. The 40 nt ssDNA substrate enhanced activity similar to the 5 -overhang duplex substrate, while the 17 nt ssDNA substrate did not enhance ATPase activity above the background (Figure 1 B). Interestingly, a 40 nt ssRNA substrate with the same sequence, but phosphorothioated at a pproximatel y e v ery 5th position (Supplementary Table S1) and a 17 nt RNA did not enhance ATPase activities, suggesting the ATPase activity of CasDinG is pr efer entially enhanced by DNA.
To evaluate whether CasDinG prefers to hydrolyze a specific ribonucleotide triphosphate we compared the release of inorganic phosphate from ATP, GTP, CTP, and UTP (Figure 1 C) in the presence of a 40 nt ssDNA. CasDinG hydrolyzed GTP and UTP, but at a rate f ourf old lower than ATP, and CTP was not hydrolyzed, indicating CasDinG pr efer entiall y hydrol yzes ATP.
To further evaluate how nucleic acid enhances CasD-inG ATPase activity, we collected ATP hydrolysis velocities with or without a constant concentration of 40 nt ssDNA, and at different concentrations of ATP. Velocity values were then fit to the Michaelis Menten equation (Figure 1  Just as the presence of nucleic acid can enhance the ATPase activity of helicases, helicase affinities for nucleic acid substrates are sometimes enhanced by the presence of ATP (36)(37)(38). To investigate if ATP enhances the ability of CasDinG to bind nucleic acid substrates we determined the dissociation constant ( K d ) of binding of our 40 nt. long ssDNA substrate to CasDinG with and without the non-hydrol yzable analo g AMP-PNP (Figure 1 E). Cas-DinG bound to the ssDNA with a K d of 25 ± 3.1 nM without AMP-PNP, and with a K d of 26 ± 2.9 nM with AMP-PNP, demonstrating the presence of a nucleotide triphosphate does not enhance nucleic acid binding. Collecti v ely, these da ta indica te CasDinG is an ATP hydrolase that is activ ated b y ssDNA. Howe v er, the general affinity of Cas-DinG for nucleic acids is not influenced by the presence of nucleotides.

CasDinG is an ATP-dependent 5 -3 DNA translocase that unwinds DN A / DN A and RN A / DN A duple x es
It remained unclear if CasDinG ATPase activity was coupled to nucleic acid translocation and duplex unwinding. To determine if CasDinG possesses helicase activity we combined CasDinG with various DNA duplexes in the presence of ATP and Mg 2+ . One strand of each duplex was 5 -endlabeled with fluorescein (FAM) to visualize strand displacement with an electrophoretic mobility shift assay (EMSA) on a polyacrylamide gel. We observed that CasDinG displaced the FAM-labeled strand from DNA duplexes with 5 -ov erhangs, but not duple xes with a 3 -ov erhang or b lunt ends, consistent with a 5 -3 unwinding polarity (Figure 2 A). Additionall y, CasDinG displaced RN A from RN A / DN A hybrid duplexes with 5 -DNA overhangs, but CasDinG did not displace DNA or RNA from duplexes with 5 -RNA overhangs, consistent with a pr efer ence for translocation on ssDNA (Figure 2 B).
The unwinding activities of CasDinG were only observed in the presence of ATP and wer e impair ed by EDTA, indicating unwinding is coupled to ATP hydrolysis and the presence of a divalent metal. To confirm ATP hydrolysis is coupled to unwinding we examined helicase activity in the presence of ADP and non-hydrol yzable analo gs ATP ␥ S and AMP-PNP. Only in the presence of ATP was duplex unwinding observed above 20% (Supplementary Figure S3A and B). To determine which divalent metals activate CasD-inG helicase activity, we examined helicase activity with a v ariety of div alent metals. We observed that Mg 2+ , Mn 2+ , Ca 2+ , Ni 2+ and Co 2+ , allow for helicase activity, whereas Zn 2+ and Cu 2+ did not (Supplementary Figure S3C). Collecti v ely these data demonstrate that CasDinG is an ATPand divalent metal ion-dependent helicase that pr efer entially translocates in the 5 -3 direction on ssDNA strands and displaces DNA or RNA complements.

CasDinG structure consists of a helicase core with three accessory domains
CasDinG shares less than 22% sequence identity and 31% sequence similarity with S. aureus and E. coli DinG (Supplementary Figure S1). The highest similarity r egions ar e the SF2 helicase motifs (Q, I, II, III, IV , V , VI) within the two RecA-like domains of the helicase core, while the least similar r egions r eside within the pr edicted accessory domains (Supplementary Figure S1). We hypothesized that amino acid differences in the accessory domains might influence the function of CasDinG in type IV-A immunity. To better understand the function of the accessory domains we determined the crystal structure of CasDinG from P. aeruginosa strain 83 at 2.95 Å resolution with an Rwork / Rfree of (18.5 / 21.5) in space group P 6 5 ( Figure 3 and Table  1 ). We solved the structure by molecular replacement using an AlphaFold2 model of N-terminally truncated CasDinG (25)(26)(27). Notably, the domains of the AlphaFold2 prediction aligned to our final model with RMSDs < 1 Å (Supplementary Figure S4). Howe v er, the positioning of the acces-sory domains was slightly different between the AlphaFold2 model and our final structure.
The CasDinG structure re v eals a SF2 helicase core of two RecA-like helicase domains (HD1 and HD2) and two accessory domains inserted within HD1 (vFe / S and arch domain) (Figure 3 A). An N-terminal domain of 103 amino acids was not observed in the electron density. Similar to other SF2 helicases, the conserved helicase motifs decorate the cleft between HD1 and HD2 ( 37 ) (Supplementary Figure S5). Alignment of the RecA helicase domains of Cas-DinG with E. coli DinG bound to ADP-BeF and ssDNA (PDB: 6FWS) re v eals high similarity within the helicase core and suggests CasDinG relies on the conserved helicase motifs to bind and hydrolyze ATP with a mechanism similar to E. coli DinG (Figure 3 C and D) ( 15 ). Additionally, se v eral residues linked to XPD disease sta tes tha t lie outside the conserved helicase motifs (e.g. R196, R614, and R706) are structurally conserved (Supplementary Figure S1 and Supplementary Figure S6) (39).
Recent structures of E. coli DinG bound to ssDNA were determined in the absence (PDB:6FWR 'open conforma-tion') and presence (PDB:6FWS 'closed conformation') of an ATP analog, re v ealing two distinct conforma tions tha t suggest a two-step mechanism for DinG-mediated DNA translocation and duplex unwinding ( Supplementary Figure S7) (15). In the first step, ATP binding causes HD2 to slide along the ssDNA in the 5 -3 direction towards HD1, while the ssDNA is held in place by HD1. The increased proximity of the RecA domains brings the two halves of the ATPase acti v e site together, promoting ATP hydrolysis. In the second step, ATP hydrolysis and ADP release  Figure S7) ( 15 ).
To determine what our CasDinG structure could re v eal about the unwinding mechanism of DinG-like helicases, we aligned HD1 of CasDinG with HD1 of the E. coli DinG binary structure or the 'open' conformation (rmsd of 3.5 Å ) (Supplementary Figure S7C). The alignment re v ealed that the HD domains of CasDinG are in a wider, or 'extra open', conformation than the HD domains of the binary E. coli DinG structure (Supplementary Figure S7D). The extra open conformation may be a crystallo gra phic artifact, or the result of having no ssDNA bound in the CasDinG structure. The second possibility implies ssDNA binding would bring HD1 and HD2 closer together to adopt a conformation like that observed in the binary E. coli DinG structure.
Similar to DinG and XPD-family helicases, CasDinG contains two accessory domains inserted within HD1. Although the two inserts in CasDinG have low sequence similarity to E. coli DinG, they share tertiary topology (Supplementary Figure S8). The first insert (residues 195-275) consists of a four alpha-helix bundle that aligns with the E. coli DinG FeS cluster domain with an RMSD of 2.3 Å using the Coot Secondary Structure Matching tool ( 15 , 21 , 22 ).
Despite this conserved topology, CasDinG lacks three of the four cysteines observed in E. coli DinG that coordinate the FeS cluster, and no FeS cluster is observed in the electron density of the CasDinG structure ( Supplementary  Figures S1 and S8A). The FeS cluster coordination in E. coli DinG stabilizes the tertiary fold of the domain, holding ␣2 in the proximity of the loop directly downstream of ␣4 (Supplementary Figure S8A). In CasDinG, the lack of a stabilizing FeS cluster is compensated for by a salt bridge formed between residues R204 and D269 (Supplementary Figure S1). These salt bridge residues appear to be fairly conserved in CasDinG sequences but not in chromosomal DinG ( 17 ). Thus, to distinguish the CasDinG domain from DinG accessory domains that coordinate FeS clusters, we named this domain in CasDinG a vestigial FeS domain or vFeS. Additional differences between CasDinG and E. coli  Figure S8). Notabl y, anal ysis of CasDinG crystal packing suggests that the vFeS position that sterically blocks ssDNA binding is likely a crystallographic artifact, indica ting a conforma tional rearrangement of the domain that allows for ssDNA would likely be allowed in solution.
In DinG, XPD, and CasDinG, the FeS domain interfaces with the other HD1 insert called the 'arch' domain ( 34 , 39-41 ). In the E. coli DinG structures the contact between the FeS and arch domains creates a pore that ssDNA passes through to interact with HD1 and HD2. While the pore created by the vFeS and arch domains in the CasDinG structure is not large enough to allow ssDNA to pass through, the PISA server ( 42 ) calculates the interface between the CasDinG vFeS and arch domains to be a pproximatel y 720 Å 2 , larger than the FeS / arch interfaces of E. coli DinG (238 Å 2 ) and Human XPD (346 Å 2 ), and indicating a similar interface between vFeS and the arch domain would likely remain, e v en in a different conformation that forms a pore through which ssDNA can pass during unwinding.
The CasDinG arch domain (residues 352-467) consists of a four-helix-bundle and a three-stranded anti-parallel beta-sheet, sharing structural topology with the arch domain of E. coli DinG and XPD helicases ( 15 , 40 , 41 ). Indeed, the Dali server reports an RMSD of 3.3 Å between aligned Nucleic Acids Research, 2023, Vol. 51, No. 15 8125 CasDinG and E. coli DinG arch domains (Supplementary Figure S9) ( 23 ). Howe v er, the arch domain of E. coli DinG is 61 amino acids longer than the CasDinG arch and contains an extra beta-loop connecting helices ␣2 and ␣3 (Supplementary Figure S9A). When aligned along HD1 the E. coli DinG arch domain appears to be ∼12 Å longer than the CasDinG arch and is rotated 25 • away from the ssDNA binding site (Supplementary Figure S9C). The arch domain of XPD makes important contacts with other proteins in the human transcription factor IIH complex ( 40 , 43 ). Thus, it is possible that the sequence, position, and size differences of the arch domains of CasDinG and E. coli DinG could be associated with differences in pr otein-pr otein interactions.

The type IV-A system recognizes a 5 -GNAWN-3 PAM
To better understand the function of CasDinG we desired to examine structure-guided mutants with the cell-based assay we previously developed to demonstrate type IV-A plasmid clearance ( 7 ). Howe v er, we were concerned that our assay was not optimal because r ecent literatur e suggested type IV-A systems prefer a 5 -AAG-3 protospacer adjacent motif (PAM) located on the 5 -side of the target sequence ( 4 , 8 ), instead of the 5 -CTTTC-3 PAM our previous assay utilized. PAMs are small nucleotide motifs that distinguish 'non-self' from 'self' targets that reside next to the nucleic acid complements of crRNA-guided surveillance complexes (44)(45)(46)(47).
To determine what PAM sequences optimally activate the type IV-A system, we performed a plasmid curing assay in biological triplicate with a library containing all 1024 combinations of 5 nucleotides adjacent to the 5 -side of the target sequence, similar to previous studies identifying PAM pr efer ences ( 30 ) (Figur e 4 ). Transfected cells wer e grown in liquid culture under immune system-inducing conditions, harvested, and deep sequenced. The depletion of a PAM sequence, when compared to no-immune system control, indicated an activating PAM sequence. Depletion scores for each PAM base position, and a PAM wheel (Figure 4 C), were generated to re v eal PAM nucleotide preferences. The data re v ealed a depletion pr efer ence for guanine at position −5, adenosine at position −3, and adenosine or thymine at position -2. There appeared to be no preference for nucleotides at positions −1 and −4, defining a pr eferr ed consensus PAM of 5 -GNAWN-3 , where W indicates A or T. Notabl y, there also a ppears to be an anti-targeting effect when guanine or cytosine is located at position −2, and when cytosine is loca ted a t position −3. The type IV-A CRISPR repeat adjacent to the 5 -side of the spacer (selfsequence) contains a cytosine at the −3 position and guanine at the −2 position, suggesting the PAM recognition mechanism has e volv ed to av oid self tar gets while gaining a mechanism to interrogate non-self targets.
To confirm that the 5 -GNAWN-3 PAM is pr eferr ed we performed plasmid clearance assays with single target plasmids containing specific activating PAMs identified in the assay (Figure 4 D). We first tested the 5 -CTTTC -3 PAM used in our previous work. Although this PAM does not contain a pr eferr ed adenosine at the −3 position or a guanine at the −5 position we still observ ed measurab le depletion of the target strand compared to the non-target.
Howe v er, consistent with our PAM library screen, PAMs that conformed to the 5 -GNAWN-3 consensus sequence, with either a −3 adenosine, a −5 guanine, or both (e.g. 5 -GGAAA-3 ) showed the strongest target clearance. Collecti v ely, these data suggest that the reason the 5 -CTTTC-3 PAM w ork ed previously is because it did not contain antitargeting bases G-C or C in the −2 or −3, position. Howe v er, other PAMs that conform to the 5 -GNAWN-3 sequence are preferred.

CasDinG accessory domains are essential for type IV-A immunity
After identifying the optimal PAM for the type IV-A system, we used our cell-based assay to investigate the role of the CasDinG accessory domains in type IV immunity. Using our structure as a guide, we mutated the plasmid encoding CasDinG to remove regions encoding the N-terminal, vFeS, or arch domain (Supplementary Figure S10). Additionally, to compare CasDinG function to other DinG-like proteins, we replaced the loop of the vFeS cluster positioned in the RecA domain cleft with the sequence observed in the FeS of DinG, we mutated Walker B residues involved in ATP hydrolysis (D337A, and E338A), and mutated two residues outside the conserved helicase motifs known to cause XPD-related disease states (R196H and R706W) ( 39 ). We then examined how these mutant CasDinG proteins cleared a target plasmid with a 5 -GGAAA-3 PAM in a type IV-A immune system assay.
Pre vious wor k showed mutations of respecti v e Walker B and R706 amino acids in XPD helicases disrupted both AT-Pase and helicase activities, while the R196H mutation only disrupted helicase activity ( 39 ). As expected, mutation of the Walker B motif (D337A, E338A), and mutations that cause XPD-related disease states (R196H and R706W) diminished type IV-A immunity (Figure 4 E), suggesting ATPmediated helicase activities are essential to CasDinG's role in type IV-A immunity. In addition to these disrupti v e point mutants, deletion of the N-terminus, the vFeS domain, and the arch domain all impaired immune system function as did mutation of the vFeS loop (Figure 4 E), indicating each of these features plays an essential role in type IV-A immunity.
To better understand why domain deletions impaired type IV activity, we recombinantly expressed and purified domain deletion CasDinG proteins. While N-terminal and arch domain deletion mutants expressed and purified at wild-type le v els, the vFeS deletion and vFeS loop mutant expressed poorly and could not be purified a t suf ficient quantities for downstream biochemical analysis. Low expression of these proteins could explain the impaired immunity phenotype observed with these mutants. To determine the stability of the mutants w e w ere able to express and purify at wild-type le v els, we performed circular dichroism to identify any major changes in the secondary structure of mutants compared to wild-type. Our results indicated similar le v els of secondary structure elements for the mutants as wild-type (Supplementary Figure S11), indicating the mutations did not disrupt overall protein folding or stability.
We ne xt e xplored how the loss of the arch and N-terminal domains influenced CasDinG enzymatic activities with the same assays we used to characterize wild-type CasDinG ATPase, nucleic acid binding, and DNA unwinding activities. (Figure 5 and Supplementary Figure S12). Removal of the Arch domain impaired ssDNA binding, and disrupted helicase activity, but did not impair ATP hydrolysis activity a t ssDNA sa tura ting conditions, consistent with a role in DNA binding and unwinding nucleic acid duplexes. In contrast, the removal of the N-terminal domain did not impair the ATPase, ssDNA binding, or helicase activities, although removal of this domain impaired in vivo immunity. Thus, the essential role of the N-terminal domain must lie outside the canonical XPD / DinG helicase mechanisms.

The N-terminal domain is predicted to adopt a dsDNA binding fold
To better understand the function of the CasDinG Nterminal domain we predicted its molecular structure with the AlphaFold2 program on the ColabFold server ( 27 ). The program predicted a compact globular fold containing three ␣-helices and two anti-par allel ␤-str ands arr anged in a knot-like configuration ( Figure 6 ). This same protein fold was predicted for se v eral CasDinG homologs, including CasDinG from other type IV-A systems that have been characterized in vivo or in vitro (e.g. Pseudomonas oleovor ans and Ar omatoleum ar omaticum ) ( 6 , 8 ). Howe v er, CasDinG pr oteins fr om Klebsiella pneumonia type IV-A systems found on lncHI1B / lncFIB plasmids appear to lack an equivalent N-terminal domain ( 48 ).
A Dali search with the predicted N-terminal domain structur e r e v ealed similarities to se v eral nucleic acid binding proteins including bacteriophage encoded transcription regulators (49)(50)(51), the dsDNA binding VirC protein involved in DNA transfer into plants from Agrobacterium ( 52 ), and bacterial defense proteins such as DndE from the DNA phosphorothioate defense system ( 53 , 54 ) and WYL1 of the CRISPR VI-D system ( 55 ). The CasDinG Nterminal domain aligned with highest Z scores to se v eral structures of a P22 phage transcriptional regulator called the ARC r epr essor. Nati v ely, the r epr essor is a homodimer of two ribbon-helix-helix peptides, but the highest similarity scor e ( Z -scor e 6.6) was to an engineered version of the ARC r epr essor that linked the two peptides together into a single chain ( 50 ). DndE and WYL proteins adopt a similar fold as the engineered linked P22 repressor. This arrangement tucks the N-terminus of the peptide underneath a loop, forming a knot-like fold observed in another protein with structural homology to the N-terminal domain with an unknown function ( 56 ) (Figure 6 A and B).
Although the predicted CasDinG N-terminal domain shares tertiary homology with se v eral dsDNA binding proteins, their amino acid sequences are not well conserved beyond the common placement of positi v ely charged residues on one side of the domain (Figure 6 B). A structure of the ARC r epr essor bound to dsDNA shows that these positi v ely charged residues interact with the backbone of DNA (Figure 6 C). Alignment of the predicted N-terminal domain with the ARC r epr essor bound to DNA r e v eals the predicted N-terminal domain has a positi v ely charged track of amino acids that could interact directly with the major groove of dsDNA in a similar fashion to the P22 ARC r epr essor, suggesting the N-terminal domain may interact with dsDNA (Figure 6 C).

DISCUSSION
CasDinG is an essential component of the type IV-A CRISPR Cas system, which clears bacteria of invasi v e nucleic acid and silences gene expression ( 7 , 8 ). The type IV-A multi-subunit complex or Csf complex is presumed to bind to DNA targets, similar to the type I Cascade complex, and then recruit the CasDinG helicase onto the resulting R-loop ( 6-8 ) (Figure 7 A). This presumption is supported by (i) recent work in Pseudomonas oleovor ans demonstra ting type IV-A systems silence LacZ expression when targeting the gene on either the coding or non-coding strand ( 8 ), and (ii) work pr esented her e demonstrating CasDinG is a DNA, but not an RNA, translocase with dsDNA and RNA / DNA hybrid helicase activity.
Previous bioinformatic and cell-based assays suggested the Csf complex prefers to target sequences downstream of a 5 -AAG-3 PAM ( 4 , 8 ). Here, we used a target library to further define the PAM pr efer ence for DNA targeting as a  The type IV-A multi-subunit Csf crRNA-guided complex binds complementary dsDNA forming an R-loop to which CasDinG is recruited. Cas-DinG unwinds the dsDNA in a 5 -3 manner, possibly distorting DNA structure and / or displacing RNA primers. The N-terminal domain of Cas-DinG likely interacts with dsDNA, but the exact role of the domain in type IV-A immunity remains unknown. 5 -GNAWN-3 PAM with a strong resistance to targeting self sequences containing a cytosine in the -3 position or guanine or cytosine in the −2 position of the PAM. Notably, the pr eviously r eported 5 -AAG-3 PAM conforms to this broader description. Still, some individually tested PAMs in the P. oleovorans system that fit our proposed consensus sequence were shown to be non-targeting ( 8 ). This observation could be due to unfavored bases in the −5 position that were not considered, or species-specific differences in the type IV-A PAM recognition mechanism. It should also be noted that our assay only looked as far as the −5 position. It remains possible that other positions further downstream contribute to the self-vs-non-self PAM recognition mechanism.
Different class 1 multi-subunit CRISPR systems use distinct PAM recognition mechanisms to distinguish self from non-self targets ( 45 ). The type III systems, which target ss-RN A, predominantl y use a self-exclusion mechanism that inactivates interference mechanisms when the target RNA base pairs with the repeat region of the crRNA-guide ( 57 ).
Alternati v ely, Type I systems use a protein-mediated mechanism to bind specific dsDNA PAM sequences ( 47 ). Here, we observe that the type IV-A PAM targeting mechanism is biased against non-complementary bases at specific positions (e.g. −2 Guanine is not complementary to the cr-RNA repeat, but is selected against), and prefers noncomplementary bases at others (e.g. −5 Guanine is preferr ed). These pr efer ences support a protein-mediated type IV-A PAM recognition mechanism that does not rely on base pairing with the crRNA repeat, similar to the dsDNA targeting type I CRISPR system ( 47 ).
DNA binding by a crRNA-guided Csf-complex will displace the non-target strand forming an R-loop (Figure 7 A). In type I CRISPR systems, the Cas3 helicase-nuclease is recruited to the R-loop formed by the Cascade complex. Once loaded onto the non-target DNA strand Cas3 uses metal-dependent ATP binding and hydrolysis to unwind and translocate in a 3 -5 direction from the target site, while the nuclease domain degrades displaced ssDNA (58)(59)(60). The data presented here suggest that CasDinG may play a similar role in type IV-A systems. Like Cas3, Cas-DinG is an ATP and metal-dependent DNA helicase. Although CasDinG unwinds with the opposite polarity as Cas3 (5 -3 instead of 3 -5 ), the helicase activity of CasD-inG would still allow CasDinG to travel from the site of Csf complex targeting along the dsDNA (Figure 5 B). All muta tions tha t impaired in vitro CasDinG helicase activity also impaired in vivo type IV-A immunity, suggesting the immune system relies on helicase activity for proper function, and consistent with recent work by Guo et al. demonstra ting tha t gene silencing by the Pseudomonas oleovorans type IV-A system appears to deplete RNA transcripts away from the site of Csf complex targeting ( 8 ). While it is possible that ATP binding and hydrolysis could regulate a yet-to-be-discovered CasDinG function, these lines of evidence strongly suggest that CasDinG ATP-dependent DNA translocase activity coupled to dsDNA unwinding and / or displacement of RNA primers is an essential activity to type IV-A immunity (Figure 7 ).
We demonstra ted tha t the accessory N-terminal, vFeS, and arch domains are essential to type IV-A immunity (Figure 4 E). Recombinant protein expression and in vitro assays indica ted tha t the removal of the vFeS cluster domain and mutation of the vFeS loop decreased protein expression and stability, while the removal of the arch impaired ssDNA binding and helicase activity. Consistent with these observations, biochemical work with XPD and Rad3 helicases from the same family as CasDinG demonstrated that