Bacterial Hachiman complex executes DNA cleavage for antiphage defense

Cui, Yongqing; Dai, Zhikang; Ouyang, Yufei; Fu, Chunyang; Wang, Yanjing; Chen, Xueting; Yang, Kaiyue; Zheng, Shuyue; Wang, Wenwen; Tao, Pan; Guan, Zeyuan; Zou, Tingting

doi:10.1038/s41467-025-57851-1

Download PDF

Article
Open access
Published: 17 March 2025

Bacterial Hachiman complex executes DNA cleavage for antiphage defense

Nature Communications volume 16, Article number: 2604 (2025) Cite this article

11k Accesses
17 Citations
2 Altmetric
Metrics details

Subjects

Abstract

Bacteria have developed a variety of immune systems to combat phage infections. The Hachiman system is a novel prokaryotic antiphage defense system comprising HamA and HamB proteins, which contains the DUF1837 and helicase domains, respectively. However, the defense mechanism remains only partially understood. Here, we present the cryo-electron microscopy (cryo-EM) structure of the Hachiman defense system featuring a fusion of Cap4 nuclease domain within HamA. Further structure analysis indicates that the DUF1837 domain on HamA resembles the PD-(D/E)XK nuclease but lacks active sites. Bioinformatics analysis reveals that catalytically inactive DUF1837 domains often recruit other functional domains to fulfill anti-phage defense. HamA interacts with HamB to form a heterodimer HamAB to mediate ATP hydrolysis and execute DNA cleavage, thus implementing antiphage defense. Our findings elucidate the structural basis of the Hachiman defense complex, highlighting the critical roles of the helicase and nuclease in prokaryotic immunity.

A DNA-gated molecular guard controls bacterial Hailong anti-phage defence

Article 30 April 2025

Phage-encoded factor stimulates DNA degradation by the Hna anti-phage defense system

Article Open access 18 May 2026

Phages overcome bacterial immunity via diverse anti-defence proteins

Article 22 November 2023

Introduction

Bacteriophages, as viruses specifically infecting bacteria, constitute the most abundant organisms on Earth^1,2. To proliferate in phage-rich environments, bacteria have evolved multiple defense mechanisms to counteract phage infection^3,4,5. Phage defense systems in bacterial genomes are frequently clustered together and termed as “defense islands”⁶. In recent years, more than one hundred novel bacterial anti-phage systems have been identified, such as Thoeris, cyclic oligonucleotide-based anti-phage signaling system (CBASS), retron, Gabija, RADAR, and pAgo^{7,8,9,10,11,12,13,14,15,16}. These systems employ various strategies to inhibit the phage reproduction cycle, such as nucleic acid degradation, nucleotide modification, and membrane disruption^{17,18,19,20,21,22,23,24,25,26,27}. Bacterial defense systems mainly including CBASS system, Avs system, Type III CRISPR system, and Lamassu system, all of which commonly utilize nucleases to combat invasive genetic elements, such as phages and plasmids, during the immune response process^9,12,28,29. These nucleases tend to be part of large protein complexes with a variety of enzymatic activities^{9,26,27,28,29,30,31,32,33,34,35,36}. Helicases and nucleases always work together to establish a cooperative defense against phage attacks^{31,36,37,38,39,40,41,42,43,44}. Nhi, a nuclease-helicase protein, confers immunity against diverse staphylococcal phages and prevents phage DNA accumulation in cells⁴⁵. The helicase/nuclease Hna provides defense against phages in Sinorhizobium meliloti⁴⁶. The SF2 helicase/nuclease Cas3 can interfere virus in type I CRISPR-Cas systems^47,48. However, the molecular basis of these defense systems containing nuclease and helicase domains remains to be fully explored.

The Hachiman system is a bacterial defense system, and it is distributed in ~4% of bacterial genomes encompassing nearly 2000 species⁷. This defense system comprises two genes: hamA, encoding a protein with a domain of unknown function (DUF1837), and hamB, encoding a protein with a predicted helicase domain. Based on bioinformatics analysis, Payne et al. classified the Hachiman systems that contain only HamA and HamB as type I, while 3.8% of Hachiman systems that include HamA, HamB, and HamC (DUF3223) were referred to as type II⁴⁹. The Hachiman system has been reported to provide broad anti-phage defense functions^7,38,49,50. For example, the Hachiman operon from Bacillus cereus B4087 is expressed in Bacillus subtilis, conferring defense against three major families of Bacillus phages⁷. However, the related molecular mechanisms of Hachiman systems remain incompletely understood.

In this study, we constructed a phylogenetic tree based on 1781 representative HamA sequences identified by Doron et al.⁷, classifying the type I Hachiman defense system into four distinct subtypes (type I-A to I-D). The type I-A Hachiman defense system contains only the DUF1837 domain in HamA, while the other three subtypes include an additional N-terminal domain. We determined the cryo-EM structure of the type I-B Hachiman defense system, which features a fusion of the N-terminal Cap4 nuclease domain within HamA. Structural analyzes reveal that the conserved DUF1837 domain in HamA resembles the PD-(D/E)XK nuclease, enabling the degradation of diverse DNA substrates. HamA recruits auxiliary functional domains, such as Cap4 endonuclease, for antiphage defense when its DUF1837 domain loses catalytic activity. HamB is a typical SF2 helicase with two RecA-like domains, exhibiting ATP hydrolysis activity in the presence of DNA, and this activity is significantly enhanced upon binding to HamA. HamA and HamB form a binary complex to exert antiphage activity collaboratively. Additionally, the HamAB complex in the type I-A Hachiman system also exhibits nuclease activity and contributes to phage defense functions.

Results

Hachiman system provides defense against phages

To investigate the anti-phage mechanisms of the Hachiman system, we initially analyzed this system located in the region of prophage CP4-57 in Escherichia. coli K12 strain, which comprises the HamA (AbpA) and HamB (AbpB) proteins⁵⁰. HamA and HamB exhibit broad conservation across bacterial species. HamA comprises a predicted Cap4 endonuclease domain and a functionally unknown domain DUF1837, and HamB contains a DEAD-box helicase domain (Fig. 1a). The coding sequences of HamA and HamB overlap by four base pairs. We introduced the entire HamAB operon (along with its native promoter) into the pACYC184 plasmid and evaluated the HamAB-mediated defense against a panel of 50 phages using the E. coli strain (MG1655 ΔhamAB). Our results show that the Hachiman system in E. coli affords 10- to 10,000-fold protection against a broad spectrum of phages from various genes. HamAB shows a strong defense phenotype against Hanrivervirus and Warwickvirus phages with a >1000-fold efficiency of plating (EOP) reduction and against T1 and Dhilonvirus phages with a >100-fold EOP reduction, whereas a less strong defense phenotype against T7 and M13 phage (Fig. 1b and Supplementary Fig. 1).

**Fig. 1: Hachiman system provides defense against diverse phages.**

To determine whether the Hachiman system functions through an abortive infection mechanism, we challenged the bacteria with phages at varying multiplicities of infection (MOIs). The Hachiman system demonstrated protection, particularly against phages T1 or TP041 at low MOIs, whereas at a high MOI, premature culture collapse occurred with the vast majority of cells infected by the initial TP041 phage attack (Fig. 1c, d), indicating that the Hachiman system operates through an abortive infection strategy, causing the death of infected cells before the maturation of phage progeny⁵¹.

Cryo-EM structure of the Hachiman complex

To elucidate the defense mechanism underlying the Hachiman system, we determined the structure of the HamA and HamB complex using single-particle cryo-electron microscopy (cryo-EM). HamA generates a 538-amino acid (aa) protein, while HamB produces a 729-aa protein (Fig. 1e). The size-exclusion chromatography analysis showed that HamA and HamB were successfully assembled into a complex HamAB through the recombinant expression and purification (Supplementary Fig. 2a). Subsequently, we resolved the cryo-EM structure of the HamAB complex at a resolution of 3.1 Å (Supplementary Fig. 2b–e).

The cryo-EM structure of the HamAB complex reveals that HamA and HamB form a stable heterodimer in a 1:1 stoichiometry, with an interface area of ~2560 Å² (Fig. 1f, g). HamA comprises an N-terminal predicted nuclease domain (residues 1–252) and a C- terminal DUF1837 domain (residues 253–538, Fig. 1e). The N-terminal domain contains a central four-stranded mixed β -sheet edged by α-helices on each side, forming a scaffold for the active site assembly (Fig. 2a and Supplementary Fig. 3a). The structure and topology of N-terminal domain is a restriction endonuclease-like fold, with catalytic residues (aspartic acid, glutamic acid, and lysine) situated in a characteristic central β strand. Structural comparisons of the N-terminal domain and the restriction endonuclease HindIII-DNA complex show structural similarities and shared catalytic residues, suggesting that HamA belongs to the PD-(D/E)XK nuclease-like superfamily (Supplementary Fig. 3b)^52,53. DALI analysis of the HamA N-terminal domain structure reveals its high similarity to the Cap4 and SeAvs3 nucleases, with Z-scores (the amount of structural similarity) of 11.9 and 10.7, respectively. These two nucleases separately serve as phage-activated DNA endonucleases in the CBASS and AVAST defense systems^9,28. Sequence alignment of HamA-Cap4 and Cap4 enzymes from CBASS systems confirms the presence of all putative active-site residues (D45, Q58 and K60) essential for metal coordination (Fig. 2b and Supplementary Fig. 3c). In agreement with these observations, alanine substitutions at the conserved active sites within the HamA Cap4 nuclease motif resulted in the complete loss of Hachiman defense activity in E. coli³⁷ (Fig. 2c, d and Supplementary Fig. 3d). These data suggest that HamA might function as a PD-(D/E)XK nuclease enzyme.

**Fig. 2: Cryo-EM structure of the HamAB complex.**

The HamB protein encompasses three distinct domains, namely, N-terminal domain, helicase core, and C-terminal helical domain (Fig. 2e). The N-terminal domain of HamB exhibits similarity to the TPR (tetratricopeptide repeat) motif (Supplementary Fig. 4a, b), and this motif is characterized by pairwise alpha-helices linked by a loop, facilitating protein-protein interactions⁵⁴. HamB, belonging to the super-family 2 (SF2) helicase family⁵⁵, contains RecA-fold domains 1 and 2 which collectively include 7 conserved helicase sequence motifs necessary for ATP hydrolysis and nucleic acid binding (Fig. 1e and Supplementary Fig. 5). The structure of the RecA domain of human RNA helicases RIG-I and MDA5 exhibit similarity to that of the RecA domain of HamB, with a root mean square deviation (RMSD) of 4.01 and 3.73 (over 168 Cα atoms), respectively (Supplementary Fig. 4c–e)^56,57. Point mutations in the conserved residues that constitute the ATP binding site (G127A/K128A/S129A) in the helicase motif I completely abolished defense activity of HamAB (Fig. 2c, d, f). Taken together, these results suggest that the ATP binding is essential for the antiphage defense of the Hachiman system³⁷.

The structure of HamAB complex reveals that HamA and HamB adopt a heterodimer conformation via extensive hydrogen bonding and salt-bridge interactions across three distinct interfaces (interfaces 1, 2, and 3) (Fig. 2g). Interface 1 includes the Cap4 nuclease domain of HamA (residues 31-34) and the TPR1 repeat (residues 31-33) of HamB. Interface 2 comprises the Cap4 domain of HamA (residues 180-251) and the RecA1 domain of HamB (residues 256-271). Finally, Interface 3 consists of the DUF1837 domain of HamA (helices α8-10, strands β5/6/9) and the TPR domain (helices 2/4/6) of HamB (Fig. 2h).

To evaluate the importance of HamA-HamB interfaces, we examined HamA-HamB interactions and phage defense function through HamAB truncations and point mutations. Consistent with our observations of the HamAB complex structure, pull-down assays show that the truncation of the DUF1837 domain in HamA completely deprives HamA of its ability to bind to HamB in vitro. Likewise, the deletion of the TPR repeats from HamB hinders the in vitro formation of the HamAB complex (Supplementary Fig. 6a). Specifically, the HamA residues R428, L432, D437, E369, E374, Y367, D433, R346, D305, D316, and R328 show hydrogen bond interactions with HamB S18/N21, N27, Y36, H56, T57/T58, N62, K66, D83, R86, Y91 and R92, respectively (Fig. 2h). Consistent with the observed interaction mode of HamAB, alanine substitutions at residues N62/K66 and Y91/R92 within the TPR repeats hinders the formation of the HamAB complex and diminishs the protection against phage infection (Fig. 2i and Supplementary Fig. 6b). These findings indicate that the N-terminal TPR domain of HamB is responsible for HamA binding and phage defense, which is in line with a recent report on Hachiman system³⁸. Mutations in the DUF1837 domain of HamA (D305A, R346A, R428A, and D433A/D437A) result in loss or decrease of phage resistance (Fig. 2i). Thus, these results highlight the essential role of the complex assembly of HamA and HamB in anti-phage defense via the Hachiman system.

ATPase activity is critical for the Hachiman function

Helicases are characterized by a set of essential conserved motifs integral to their activity. These enzymes typically comprise two RecA-like domains responsible for ATP binding and hydrolysis⁵⁵. Considering the pivotal role of ATP binding sites in the antiviral function of the Hachiman system, we performed a malachite green assay in vitro to investigate HamB’s ATPase activity. Since nucleic acid often enhances the ATPase activities of helicases, we examined ATP hydrolysis with various DNA and RNA substrates. The results indicate that HamB catalyzes the release of inorganic phosphate from ATP, with 52-bp dsDNA fragment as substrate. Conversely, substrates such as 52-nt ssDNA, pUC19 plasmid dsDNA, phage ΦX174 ssDNA, 1200 bp dsDNA, and 21-nt ssRNA fail to significantly enhance HamB’s ATPase activity. Mutation of the ATP binding sites of HamB led to a complete loss of ATPase activity (Fig. 3a). To explore the effects of HamA on HamB ATPase activity, we quantified the release of inorganic phosphate from ATP hydrolysis based on the assays of HamA, HamB, and HamAB complex, respectively. Our analysis demonstrates that the ATPase activity of HamAB complex is significantly enhanced in the presence of 52-bp dsDNA, showing an almost tenfold increase relative to that of HamB alone (Fig. 3b). In addition, the mutations of the conserved catalytic sites in HamA do not affect HamAB ATPase activity, whereas the mutation of ATP binding sites in HamB results in a complete loss of hydrolysis activity of HamAB (Fig. 3c). This indicates that ATP hydrolysis by HamB is essential for Hachiman activity, which is in accordance with our phage infection assay (Fig. 2c, d).

**Fig. 3: ATPase and nuclease activity of HamAB complex.**

The HamB structure reveals an SF2 helicase core containing two RecA-like helicase domains. To determine whether HamB possesses helicase activity, we incubated HamB with various DNA duplexes in the presence of ATP and Mg²⁺. The results show that HamB does not unwind overhang substrates or blunt dsDNA, with helicase activity undetectable (Fig. 3d). Since HamAB complex exhibits a higher ATP hydrolysis activity than HamB, we further tested DNA unwinding by HamA*B (D45A/Q58A/K60A, HamA nuclease inactive mutant) complex. In contrast, HamA*B can unwind various substrates incubated with overhang or blunt dsDNA containing a 15-bp duplex (Fig. 3e). These results demonstrate that HamA coordinates with HamB to unwind dsDNA.

Nuclease activity of the HamAB complex

Further, we evaluated the ability of HamA and HamAB proteins to cleave DNA substrates in vitro. After incubating with a dsDNA substrate, HamA exhibits no nuclease activity. In contrast, the HamAB complex effectively catalyzes the complete degradation of dsDNA into fragments smaller than 100 bp. Moreover, the mutation of conserved active sites of the HamA (D45A or K60A) eliminates nuclease activity of HamAB, indicating that the N-terminal Cap4 nuclease domain of the HamA is of cleavage capacity, but it is auto-inhibited in the full-length protein (Fig. 3f). Additionally, the nuclease function of the HamAB complex is dependent on Mg²⁺, Co²⁺, or Zn²⁺, but independent of Ca²⁺, Cu²⁺, and Ba²⁺ ions (Fig. 3g). The HamAB complex degrades a wide range of DNA, including linear dsDNA, plasmid DNA, ΦX174 ssDNA, phage genome dsDNA, and bacterial genomic DNA, as indicated by clear degradation bands (Fig. 3h). Overall, these findings suggest that the Hachiman complex adopts a Cap4 enzyme-like reaction mechanism to degrade nucleic acids.

DUF1837 is a PD-(D/E)XK nuclease domain in the Hachiman defense system

DUF1837, the C-terminal of the HamA protein, remains functionally uncharacterized. Structural analysis of HamA reveals that the DUF1837 domain comprises a central five-stranded β sheet decorated on either side by alpha helices, which resembles the core motif of the proteins in the PD-(D/E)XK phosphodiesterase superfamily (Fig. 4a). The top hit of DALI search for DUF1837 is type II restriction endonuclease PaqCI, with a Z score of 5.6. The active site core is the PD-(D/E)XK motif, essential for phosphodiester bond cleavage in nucleic acids. Our analysis reveals the absence of these active sites in the DUF1837 domain of HamA. In contrast, in a different Hachiman defense system from E. coli DEC14B, the structure (predicted by AlphaFold2) of DUF1837 domain of HamA closely resembles that of PaqCI nuclease, but DUF1837 domain includes all the active sites of PD-(D/E)XK nuclease family (Fig. 4a and Supplementary Fig. 7a)⁵⁸. Sequence alignment classifies DUF1837 domain of HamA into two categories: one with conserved active sites and the other without catalytic active sites (Supplementary Fig. 7a).

**Fig. 4: DUF1837 belongs to the PD-(D/E)XK nuclease family.**

To better understand the HamA diversity in Hachiman defense systems, we generated a phylogenetic tree based on 1781 representative sequences identified by ref. ⁷. The phylogenetic tree shows that the HamA proteins are clustered into four distinct clades. Among them, type I-A contains only the DUF1837 domain, with conserved active sites, constituting the majority clade (1539 of 1781, accounting for 86.4%), whereas type I-B, type I-C, and type I-D are characterized by an additional N-terminal domain apart from the DUF1837 domain (Fig. 4b, c). This additional domain contains predicted Cap4 nucleases, peptidases, or other unidentified domains (hereafter referred to as “X”), with its average length ranging from 87 to 577 amino acids (Fig. 4b, d). We observed that the PD-(D/E)XK sites are absent in the DUF1837 domains of type I-B, type I-C, and type I-D, based on which we speculated that these DUF1837 domains might be catalytically inactive, functioning as scaffolds.

To assess the protective capacity of type I-A Hachiman systems against phage infection, we investigated the defense activity of the HamAB complex (from E. coli strain DEC14B) comprised of HamA containing only a DUF1837 domain and HamB containing a helicase domain (Fig. 4e). We cloned the HamAB operon along with its native promoter into plasmid pACYC184, and subsequently introduced plasmid pACYC184 into E. coli MG1655 ΔhamAB, followed by challenge with various E. coli phages. The results show that the type I-A Hachiman system provides a protective effect against seven distinct dsDNA bacteriophages, including model phages T4 and T7 (Supplementary Fig. 7b, c). In addition, the mutation of the active sites E150A and K152A in HamA results in a loss of functionality, suggesting that the nuclease activity of the DUF1837 domain is crucial for this defense. A point mutation (G272A/K273A/T274A) of the helicase domain in HamB impedes ATP binding, thereby eliminating the protective effect of the Hachiman system (Fig. 4f).

Given that the DUF1837 domain exhibits structural homology with type II restriction endonucleases, we hypothesize that HamA DUF1837 is a PD-(D/E)XK nuclease family member. To evaluate the nuclease activity of the type I-A Hachiman system in vitro, HamA and HamB were recombinantly expressed and purified from E. coli. The co-expression and purification experiments confirm the formation of HamAB complex (Supplementary Fig. 7d). Incubation of HamA and HamAB with linear double-stranded DNA showed progressive substrate degradation only in the presence of HamAB, which is in line with the observation of the type I-B Hachiman system from E. coli K-12. The nuclease activity of HamAB is dependent on the catalytic residues of HamA-DUF1837, and it is abolished by the E150A/K152A nuclease mutation in HamA, which is consistent with results of phage spot test (Fig. 4f, g). Further, we investigated the substrate specificity of type I-A Hachiman system and found that HamAB effectively degraded various DNA substrates, including 1200-bp double-stranded DNA (dsDNA), supercoiled pUC19 plasmid DNA, ΦX174 ssDNA, phage genome dsDNA, and MG1655 genome DNA, yielding the fragments ~100 nucleotides in length. This result suggests that HamAB has a non-specific nuclease activity (Fig. 4h). Taken together, these results collectively support the hypothesis that the DUF1837 domain acting as a PD-(D/E)XK-like nuclease is essential for phage defense in the type I-A Hachiman system.

Discussion

The Hachiman system provides protection against phage infection in host cells through an abortive infection mechanism. Here, we investigated bacterial type I-A and type I-B Hachiman defense systems, revealing that both subtypes have broad-spectrum anti-phage functions, which are mediated by two proteins, namely, HamA with DNA endonuclease activity and HamB as an SF2-type helicase. All HamA proteins possess a DUF1837 domain. Structural analysis reveals that this domain is part of the PD-(D/E)XK nuclease family. Specifically, in the type I-A Hachiman system, the DUF1837 domain includes all conserved catalytic sites essential for its function. In contrast, in the type I-B, I-C, and I-D systems, the DUF1837 domain displays a structure similar to the PD-(D/E)XK nuclease, but it lacks catalytic sites and is integrated with other domains to act as effector proteins, which is crucial for phage resistance. Our data suggest that HamA alone exhibits no nuclease activity in the presence of diverse nucleic acid substrates. Likewise, HamB alone does not exhibit helicase activity, either. However, when HamA binds with HamB to form the HamAB complex, this complex undergoes DNA unwinding through ATP hydrolysis, exerting DNA degradation activities. A recent study by Tuck et al. showed that HamA and HamB, classified as part of the type I-A Hachiman system, can form a heterodimeric nuclease-helicase complex. Upon detecting DNA damage, the activated Hachiman system degrades both phage and host DNA³⁸. These results collectively indicate that the formation of the HamAB complex is crucial for resistance against phage infection (Fig. 4i).

Tuck et al. indicated that HamB from the type I-A system can detect DNA damage, recognize and bind to 3′ ssDNA ends to trigger ATPase activity, further activating HamA to exert nuclease activity³⁸. However, our data suggest that the ATPase activity of HamB in the type I-B Hachiman system is significantly activated by dsDNA. Phylogenetic analysis shows that type I-A and type I-B HamB occupy distinct branches, indicating significant sequence divergence (Supplementary Fig. 8a). Structural analysis reveals that while both subtypes share conserved DNA-binding amino acids, type I-B lacks the winged-helix (WH*) and oligonucleotide-binding (OB) fold domains present in type I-A (Supplementary Fig. 8b, c)^59,60. The OB-fold domain, crucial for binding single-stranded nucleic acids, is absent in type I-B, suggesting differences in substrate specificity^61,62. These structural and sequence variations likely explain why the ATPase activity of the two subtypes is triggered by different substrates.

Our findings indicate that the loss of nuclease activity in the effector DUF1837 domain prompts the Hachiman immune system to recruit an auxiliary effector to sustain antiphage defense. Furthermore, we hypothesize that the DUF1837 domain may function as a scaffold for assembling a multiprotein complex containing active homologs. This hypothesis is supported by our observation that the DUF1837 domain of HamA facilitates the assembly of the HamAB complex. Additionally, our data demonstrate that the ATP hydrolytic activity of the HamAB complex is significantly higher than that of HamB alone (Fig. 3b), aligning with recent results³⁸. However, a discrepancy exists between the two studies: in their research, the mutation of the nuclease active sites in HamA of the type I-A Hachiman system markedly influences HamAB ATP hydrolysis activity³⁸. In contrast, the mutation of Cap4 nuclease active sites in HamA of the type I-B Hachiman system does not affect the ATP hydrolysis activity of HamAB (Fig. 3c). This suggests that the inactive DUF1837 domain acts as an intermediate to mitigate direct influence between the two catalytic domains, thereby significantly increasing the probability of DUF1837 fusing with other domains, such as peptidases, ultimately broadening the diversity of immune mechanisms in bacteria. Similarly, the prokaryotic Argonaute immune system employs various effector domains for antiviral defense due to the absence of nuclease activity in its PIWI domain^11,63. Based on this, we speculate that bacteria adopt this strategy to enhance their defensive arsenal through long-term evolution against phages.

Methods

Bacterial strains, phages, and plasmids construction

The full-length of type I-B hamAB genes were amplified from the Escherichia coli str. K-12 (2761204–2765368 of GenBank Accession U00096.3). The full-length of type I-A HamAB genes from Escherichia coli DEC14B (67654–71936 of GenBank Accession AIHL01000073.1) were synthesized. For the construction of HamAB complex plasmids: (1) The hamA was subcloned into a pET15D vector (Novagen) between the NdeI and XhoI restriction sites, resulting in a fused protein with an N-terminal 6× His tag. The HamB gene was subcloned into the pBB75 vector (Novagen) without any tag between the NdeI and EcoRI restriction sites. (2) The Type I-A hamA gene was subcloned into the pBB75 vector (Novagen) without any tag, and the hamB was subcloned into a pET21b vector (Novagen) with a C-terminal 6×His-tag between the NdeI and EcoRI restriction sites. Plasmids encoding HamA and HamB were co-transfected in E. coli strain BL21(DE3). For the construction of individual HamA and HamB plasmids: The HamA was subcloned into a pET21b vector (Novagen) with a C-terminal 6×His-tag; The HamB was subcloned into a pET15D vector (Novagen) with an N-terminal 6×His-tag. HamA and HamB mutants were amplified by Fusion PCR. Plasmids encoding HamA or HamB were transfected in E. coli strain BL21(DE3). The Hachiman genetic unit with its native promoter was amplified from its native host and subcloned into pACYC184 vectors for the in vivo experiments (plasmids used in the study see Supplementary Data 1 and primer used for cloning see Supplementary Data 2). Except for the model bacteriophage, the other collections were isolated from sewage samples on E. coli MG1655 (phage information see Supplementary Data 3), as previously described⁶⁴. All primers were chemically synthesized and purchased from Sangon Biotech.

Protein expression and purification

For the preparation of HamAB complex, the HamA and HamB, or the boundary and mutants, were co-expressed in E. coli strain BL21(DE3) using Lysogeny broth (LB) medium. The cells were induced by the addition of isopropyl-β-D-thioacetamide (final concentration of 200 μM) when OD₆₀₀ reached 1.1, and induction was performed at 16 °C for 16 h. For HamAB complex (from the E. coli K-12 and E. coli DEC14B), HamA protein (from the E. coli DEC14B), and HamB protein (from the E. coli K-12) purification, the harvested cells were resuspended in lysis buffer containing 25 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The lysate was disrupted using a homogenizer (JNBIO), and the cell debris was removed via centrifugation at 20,000 × g for 1 h at 4 °C. The target protein was purified over Ni²⁺ nitrilotriacetate affinity resin (Ni-NTA, Qiagen) and eluted with Elution buffer (25 mM Tris-HCl pH 8.0, 250 mM imidazole). The eluted protein was further purified using ion-exchange chromatography (Source 15Q 10/100, GE Healthcare). The purified protein was concentrated and subsequently purified by gel filtration chromatography (Superose 6 10/300; GE Healthcare) in SEC buffer (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM dithiothreitol). The purity of the proteins was examined by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie blue staining. The mutant proteins were purified similarly to the wild-type proteins. For the HamA protein (from the E. coli K-12) purification, the collected cells were resuspended in lysis buffer (25 mM Tris-HCl, pH 8.0, 1.5 M NaCl) and subsequently disrupted by pressure homogenizer at 4 °C. The target protein was purified over Ni²⁺ affinity resin, Source 15Q10/100, and Superose 6 10/300 columns used in tandem. The Elution buffer and SEC buffer of purified HamA remains consistent with the above.

Cryo-EM grid preparation and data acquisition

For cryo-EM data acquisition of the HamAB complex (E. coli K-12), 3.5 µl sample was dropped onto glow-discharged holey carbon grids (Quantifoil Au R1.2/1.3, 300 mesh). After blotting for 4.5 s with 100 % humidity at 8 °C, the grids were plunge-frozen in liquid ethane cooled by liquid nitrogen in Vitrobot Mark IV (ThemoFisher Scientific). Subsequently, the cryo-grids were screened using 300 kV Titan Krios electron microscopes (Thermo Fisher Scientific) and equipped with a Gatan K3 Summit Detector positioned to post a GIF Quantum Energy Filter (slit width 20 eV). Images for the HamAB were recorded in the counting mode with a magnification of 81,000× and collected with a pixel size of 1.07 Å and a defocus range of −1.2 to −2.2 μm. Each micrograph stack, which contains 32 frames, was exposed for 3.5 s with a total electron dose of 50 e⁻Å⁻². Preliminary data processing results show that the HamAB exhibit strong preferential orientation on cryo-EM grids. Therefore, 6881 and 4864 cryo-EM micrographs were collected at 0° and 30° specimen tilt, respectively⁶⁵. EPU software was used for fully automated data collection.

Cryo-EM data processing

A diagram of the procedures for data processing is presented in Supplementary Fig. 2. The movies collected at tilt angles of 0 and 30 degrees are separately motion corrected by Motioncor2 with 7 by 7 patches and CTF estimated by cryoSPARC patch CTF estimation⁶⁶. From the datasets at 0° and 30°, 5752 and 3062 micrographs, respectively, were manually selected and merged into a total of 8814 micrographs for subsequent processing. A total of 3,516,428 particles were automatically picked using cryoSPARC blob picker, and the particle picking was optimized by Topaz software^66,67. The particles underwent multiple rounds of 3D classification. In brief, the particles were extracted into a box of 240 Å and then binned by 4, 2, and 1, respectively, facilitating the 3D classification. Following the 3D classification, the well-classified particles were merged, deduplicated, and re-extracted. From the classification results of the original-sized particle images, 440,333 particles (~26.4% of the total) were selected for 3D refinement, resulting in a cryo-EM density map with an overall resolution of 3.1 Å based on gold-standard Fourier shell correlation (FSC)⁶⁸. The local resolution of the map was estimated to be 2.8 to 4 Å using Resmap (v1.1.4)⁶⁹.

Model building and refinement

The initial model of HamA and HamB predicted by AlphaFold2⁷⁰ was rigidly docked to fit into the cryo-EM density map of HamAB, resulting in the acquisition of the initial coordinates file for the HamAB complex structure. The initial model of the HamAB complex was manually adjusted using COOT⁷¹, and invisible segments were removed, yielding an optimized model that included the 7-537 segment of HamA, the 2-449 segment of HamB, and the 510-728 segment of HamB. The complex models were refined against the map using PHENIX in real space with secondary structure and geometry restraints⁷². Model quality was evaluated using the MolProbity scores⁷³, the Ramachandran plots, and EMRinger (see Supplementary Table 1)⁷⁴. Figures were generated using ChimeraX v.1.2.5 and PyMol v.2.5.1.

In vitro pull-down assay

A co-expression coupled Ni-NTA pull-down assay was performed to detect the interaction of HamA and HamB. In this system, no tag HamB and 6×His-tagged HamA (wild-type or the boundary and mutants) were co-expressed within E. coli strain BL21(DE3). Then, the cell debris of the lysed cells and supernatant were loaded on Ni-NTA beads. After washing unbound proteins, the His-tagged HamA and interacting HamB were co-eluted by using an elution buffer (25 mM Tris-HCl pH 8.0, 250 mM imidazole). Elution was analyzed by SDS-polyacrylamide gel electrophoresis and revealed key residues for the interaction between HamA and HamB.

DNA degradation assays

DNA nuclease assays were carried out on various DNA in 20 μl reaction buffer (25 mM Tris-HCl pH 8.0, 5 mM MgCl₂, and 1 mM DTT) with 80 nM protein. The reactions were performed at 37 °C for 30 min and stopped by the addition of 2 µl 6× loading buffer (60 mM EDTA, 20 mM Tris-HCl pH 7.5, 30% glycerol, 0.1% SDS). After the reactions, products were separated on a 1.0% TAE agarose gel and run at 150 V for 20 min. The Gels were visualized using a Gel Doc^TM Molecular imager (BIO-RAD). Source data are provided as a Source data file.

ATPase assay

The ATPase reactions were carried out in 20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, and 1 mM DTT. We added 500 nM or 50 nM protein in a 20 μL reaction volume containing 2 mM ATP, 50 nM or 100 ng DNA substrate at 30 °C for 30 min. The release of inorganic phosphate (Pi) was monitored by Malachite Green Phosphate Assay as previously described⁷⁵. The reaction products were measured by adding 150 µL malachite green solution (86 µL of 28 mM ammonium heptamolybdate in 2.1 M H₂SO₄ and 64 µL of 0.76 mM malachite green in 0.35% polyvinyl alcohol). After 10 min, absorption at 595 nm was monitored.

Helicase assays

To prepare substrates of double-stranded DNA (dsDNA) with 15 bp duplexes and a forked 15 nt OH, 15 nt 3′ OH, 15 nt 5′ OH, and no overhang for helicase assays, 5′ FAM-labeled single-stranded DNA (ssDNA) and unlabeled complementary ssDNA (see Supplementary Table 2 for oligonucleotide information) were mixed in a 1:2 molar ratio, denatured at 95 °C for 5 min, and annealed at 25 °C. For helicase assays, 40 nM 5′ FAM-labeled dsDNA substrates were incubated with HamB or HamA^K60AB at indicated concentrations (200 nM, 40 nM, and 8 nM) and 2.5 mM ATP in helicase buffer (20 mM Tris-HCl pH 8.0, 1 mM DTT, and 5 mM MgCl₂) supplemented with 1 μM unlabeled ssDNA (corresponding to the 5′ FAM-labeled ssDNA) to prevent reannealing. 10 μL reaction mixtures were incubated at 30 °C for 15 min, and then the unwinding reactions were stopped by adding 25 mM EDTA to the mixtures. Reactions without ATP served as negative controls. The reaction mixtures without proteins were incubated at 95 °C for 10 min as positive controls for unwinding. Samples were resolved on a 12% Tris-glycine polyacrylamide gel at 150 V for 5 h at 4 °C. The fluorescent signal of FAM-labeled DNA was detected using a Typhoon FLA-5100 scanner (Fujifilm Life Science, Japan). FAM-labeled DNA were synthesized and purchased from GenScript.

Phylogenetic analysis of HamA

According to the Hachiman systems identified in a previous study⁷, 1781 protein sequences of HamA used in this study were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/sites/batchentrez). The multiple sequence alignment was conducted using MAFFT version 7.310 with an automatic model⁷⁶, followed by automated alignment trimming using trimAl version 1.4⁷⁷. Subsequently, a phylogenetic tree based on the trimmed sequence alignment was constructed using IQ-TREE version 2.0.3 with parameters -m MFP (ModelFind plus) -bb 1000 (1000 iterations of the ultrafast bootstrap)⁷⁸. The tree was visualized using iTOL⁷⁹. Additional domains beyond DUF1837 within HamA were analyzed using the tools HHpred⁸⁰ and NCBI’s Conserved Domain Database (CCD) with default parameters⁸¹.

Bacterial liquid growth assays

Bacterial growth curves were measured in liquid media. The hamAB gene from Escherichia coli strain K-12 was cloned into the pACYC184 vector. The recombinant vectors were transformed into E. coli MG1655 (ΔhamAB) strain. An empty vector was used as a control. A single bacterial colony was picked from a fresh LB agar plate and grown in LB broth supplemented with chloramphenicol (25 mg mL⁻¹) at 37 °C. Overnight cultures were diluted 1:100 into a liquid LB medium containing chloramphenicol (25 mg mL⁻¹) and grown at 37°C for 2 h to an OD₆₀₀ of about 0.18–0.2. Subsequently, MG1655 (ΔhamAB) strain harboring the hamAB gene cassette or the empty vector was infected with phages T1 or TP041 at t = 0 at a multiplicity of infection (MOI) of 0, 0.02, or 2. Growth was monitored using a FLUOstar Omega for 6 h at 30 °C with OD₆₀₀ readings every 6 min. Graphs were produced using GraphPad Prism 8 software.

Phage spot test

The phage was propagated by picking a single phage plaque into a liquid culture of E. coli MG1655 grown at 37 °C in LB medium supplemented with 0.1 mM MnCl₂ and 5 mM MgCl₂. The culture was centrifuged for 10 min at 2000 × g, and the supernatant was filtered using a 0.45 μM filter to remove bacterial debris. The phage was then amplified and purified. The reconstructed pACYC184 plasmid was transformed into E. coli MG1655 (ΔhamAB) strain, E. coli JM109 strain (as the sensitive host bacteria for M13 phage), and DMB127 strain (as the sensitive host bacteria for ΦX174 phage). Different bacteria strains were incubated with chloramphenicol (25 mg mL⁻¹) at 37 °C. After overnight incubation, 500 μl of the bacterial culture was mixed with 4.5 ml molten top agar (containing 10 g L⁻¹ tryptone, 5 g L⁻¹ yeast extract, 10 g L⁻¹ NaCl, and 5 g L⁻¹ agar), and the entire samples were poured onto LB plates containing antibiotic. Phage solution was diluted in LB at seven 10-fold serial dilutions, namely, 10⁰–10⁻⁶. After the top agar had solidified, 2 μl drops of the diluted phage culture were spotted on the bacterial layer. Plates were incubated at 30 °C overnight and then imaged. Source data are provided as a Source data file.

Statistics and reproducibility

Statistical analyzes were performed using GraphPad Prism 8 (GraphPad Software Inc., USA). Sample sizes were chosen according to similar experiments published in previous literatures^38,39. Multi-group comparisons were determined by Two-sided Student’s t-test. All data are presented as the mean ± SD of three replicates, and p < 0.05 was considered statistically significant. All P values are indicated above the plots.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The atomic coordinates and EM density for the reported structures of the HamAB complex (PDB: 8ZUE; EMDB: EMD-60482) have been deposited in the Protein Data Bank (www.rcsb.org) and the Electron Microscopy Data Bank (www.ebi.ac.uk/pdbe/emdb/). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. Source data are provided with this paper.

References

Salmond, G. P. C. & Fineran, P. C. A century of the phage: past, present and future. Nat. Rev. Microbiol. 13, 777–786 (2015).
Article CAS PubMed MATH Google Scholar
Clokie, M. R. J., Millard, A. D., Letarov, A. V. & Heaphy, S. Phages in nature. Bacteriophage 1, 31–45 (2011).
Article PubMed PubMed Central Google Scholar
Bernheim, A. & Sorek, R. The pan-immune system of bacteria: antiviral defence as a community resource. Nat. Rev. Microbiol. 18, 113–119 (2020).
Article CAS PubMed MATH Google Scholar
Dy, R. L., Richter, C., Salmond, G. P. C. & Fineran, P. C. Remarkable mechanisms in microbes to resist phage infections. Annu. Rev. Virol. 1, 307–331 (2014).
Article PubMed Google Scholar
Hampton, H. G., Watson, B. N. J. & Fineran, P. C. The arms race between bacteria and their phage foes. Nature 577, 327–336 (2020).
Article ADS CAS PubMed Google Scholar
Makarova, K. S., Wolf, Y. I., Snir, S. & Koonin, E. V. Defense islands in bacterial and archaeal genomes and prediction of novel defense systems. J. Bacteriol. 193, 6039–6056 (2011).
Article CAS PubMed PubMed Central MATH Google Scholar
Doron, S. et al. Systematic discovery of antiphage defense systems in the microbial pangenome. Science 359, eaar4120 (2018).
Article ADS PubMed PubMed Central MATH Google Scholar
Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561.e12 (2020).
Article CAS PubMed MATH Google Scholar
Lowey, B. et al. CBASS immunity uses CARF-Related effectors to sense 3′–5′- and 2′–5′-linked cyclic oligonucleotide signals and protect bacteria from phage infection. Cell 182, 38–49.e17 (2020).
Article CAS PubMed PubMed Central Google Scholar
Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Koopal, B. et al. Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA. Cell 185, 1471–1486.e19 (2022).
Article CAS PubMed PubMed Central MATH Google Scholar
Millman, A. et al. An expanded arsenal of immune systems that protect bacteria from phages. Cell Host Microbe 30, 1556–1569.e5 (2022).
Article CAS PubMed MATH Google Scholar
Vassallo, C. N., Doering, C. R., Littlehale, M. L., Teodoro, G. I. C. & Laub, M. T. A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Nat. Microbiol. 7, 1568–1579 (2022).
Article CAS PubMed PubMed Central Google Scholar
Bernheim, A. et al. Prokaryotic viperins produce diverse antiviral molecules. Nature 589, 120–124 (2021).
Article ADS CAS PubMed Google Scholar
Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems. Nature 609, 144–150 (2022).
Article ADS CAS PubMed MATH Google Scholar
Zhang, T. et al. Direct activation of a bacterial innate immune system by a viral capsid protein. Nature 612, 132–140 (2022).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Morehouse, B. R. et al. STING cyclic dinucleotide sensing originated in bacteria. Nature 586, 429–433 (2020).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Ofir, G. et al. Antiviral activity of bacterial TIR domains via immune signalling molecules. Nature 600, 116–120 (2021).
Article ADS CAS PubMed MATH Google Scholar
Tal, N. et al. Cyclic CMP and cyclic UMP mediate bacterial immunity against phages. Cell 184, 5728–5739.e16 (2021).
Article CAS PubMed PubMed Central MATH Google Scholar
Hogrel, G. et al. Cyclic nucleotide-induced helical structure activates a TIR immune effector. Nature 608, 808–812 (2022).
Article ADS CAS PubMed MATH Google Scholar
Morehouse, B. R. et al. Cryo-EM structure of an active bacterial TIR–STING filament complex. Nature 608, 803–807 (2022).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Duncan-Lowey, B. et al. Cryo-EM structure of the RADAR supramolecular anti-phage defense complex. Cell 186, 987–998.e15 (2023).
Article CAS PubMed PubMed Central MATH Google Scholar
Gao, Y. et al. Molecular basis of RADAR anti-phage supramolecular assemblies. Cell 186, 999–1012.e20 (2023).
Article CAS PubMed MATH Google Scholar
Shen, Z. et al. Oligomerization-mediated activation of a short prokaryotic Argonaute. Nature 621, 154–161 (2023).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Gao, X. et al. Nucleic-acid-triggered NADase activation of a short prokaryotic Argonaute. Nature 625, 822–831 (2023).
Article ADS PubMed MATH Google Scholar
Antine, S. P. et al. Structural basis of Gabija anti-phage defence and viral immune evasion. Nature 625, 360–365 (2024).
Article ADS CAS PubMed Google Scholar
Li, J. et al. Structures and activation mechanism of the Gabija anti-phage system. Nature 629, 467–473 (2024).
Article ADS CAS PubMed MATH Google Scholar
Gao, L. A. et al. Prokaryotic innate immunity through pattern recognition of conserved viral proteins. Science 377, eabm4096 (2022).
Article CAS PubMed PubMed Central Google Scholar
Rostøl, J. T. et al. The Card1 nuclease provides defence during type III CRISPR immunity. Nature 590, 624–629 (2021).
Article ADS PubMed PubMed Central Google Scholar
Yang, X.-Y., Shen, Z., Wang, C., Nakanishi, K. & Fu, T.-M. DdmDE eliminates plasmid invasion by DNA-guided DNA targeting. Cell 187, 5253–5266.e16 (2024).
Article CAS PubMed Google Scholar
Loeff, L. et al. Molecular mechanism of plasmid elimination by the DdmDE defense system. Science 385, 188–194 (2024).
Article CAS PubMed MATH Google Scholar
Cheng, R. et al. A nucleotide-sensing endonuclease from the Gabija bacterial defense system. Nucleic Acids Res. 49, 5216–5229 (2021).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Cheng, R. et al. Prokaryotic Gabija complex senses and executes nucleotide depletion and DNA cleavage for antiviral defense. Cell Host Microbe 31, 1331–1344.e5 (2023).
Article CAS PubMed MATH Google Scholar
Loeff, L., Walter, A., Rosalen, G. T. & Jinek, M. DNA end sensing and cleavage by the Shedu anti-phage defense system. Cell 188, 721–733.e17 (2025).
Gu, Y. et al. Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains. Mol. Cell 85, 523–536.e6 (2025).
Bravo, J. P. K., Ramos, D. A., Fregoso Ocampo, R., Ingram, C. & Taylor, D. W. Plasmid targeting and destruction by the DdmDE bacterial defence system. Nature 630, 961–967 (2024).
Article CAS PubMed Google Scholar
Sasaki, T. et al. Phage single-stranded DNA-binding protein or host DNA damage triggers the activation of the AbpAB phage defense system. mSphere 8, e00372-23 (2023).
Article PubMed PubMed Central MATH Google Scholar
Tuck, O. T. et al. Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex. Cell 187, 6914–6928.e20 (2024).
Article CAS PubMed MATH Google Scholar
Shen, Z., Lin, Q., Yang, X.-Y., Fosuah, E. & Fu, T.-M. Assembly-mediated activation of the SIR2-HerA supramolecular complex for anti-phage defense. Mol. Cell 83, 4586–4599.e5 (2023).
Article CAS PubMed PubMed Central MATH Google Scholar
Tang, D. et al. Multiple enzymatic activities of a Sir2-HerA system cooperate for anti-phage defense. Mol. Cell 83, 4600–4613.e6 (2023).
Article CAS PubMed MATH Google Scholar
Ofir, G. et al. DISARM is a widespread bacterial defence system with broad anti-phage activities. Nat. Microbiol. 3, 90–98 (2017).
Article PubMed PubMed Central MATH Google Scholar
Bravo, J. P. K., Aparicio-Maldonado, C., Nobrega, F. L., Brouns, S. J. J. & Taylor, D. W. Structural basis for broad anti-phage immunity by DISARM. Nat. Commun. 13, 2987 (2022).
Article ADS CAS PubMed PubMed Central Google Scholar
Hu, H. et al. Structure and mechanism of the Zorya anti-phage defense system. Nature https://doi.org/10.1038/s41586-024-08493-8 (2024).
Huiting, E. & Bondy-Denomy, J. A single bacterial enzyme i(NHI)bits phage DNA replication. Cell Host Microbe 30, 417–419 (2022).
Article CAS PubMed Google Scholar
Bari, S. M. N. et al. A unique mode of nucleic acid immunity performed by a multifunctional bacterial enzyme. Cell Host Microbe 30, 570–582.e7 (2022).
Article CAS PubMed MATH Google Scholar
Sather, L. M. et al. A broadly distributed predicted helicase/nuclease confers phage resistance via abortive infection. Cell Host Microbe 31, 343–355.e5 (2023).
Article CAS PubMed Google Scholar
Brouns, S. J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Jackson, R. N., Lavin, M., Carter, J. & Wiedenheft, B. Fitting CRISPR-associated Cas3 into the helicase family tree. Curr. Opin. Struct. Biol. 24, 106–114 (2014).
Article CAS PubMed MATH Google Scholar
Payne, L. J. et al. Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types. Nucleic Acids Res. 49, 10868–10878 (2021).
Article CAS PubMed PubMed Central MATH Google Scholar
Yasui, R., Washizaki, A., Furihata, Y., Yonesaki, T. & Otsuka, Y. AbpA and AbpB provide anti-phage activity in Escherichia coli. Genes Genet. Syst. 89, 51–60 (2014).
Article CAS PubMed Google Scholar
Lopatina, A., Tal, N. & Sorek, R. Abortive infection: bacterial suicide as an antiviral immune strategy. Annu. Rev. Virol. 7, 371–384 (2020).
Article CAS PubMed Google Scholar
Steczkiewicz, K., Muszewska, A., Knizewski, L., Rychlewski, L. & Ginalski, K. Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily. Nucleic Acids Res. 40, 7016–7045 (2012).
Article CAS PubMed PubMed Central Google Scholar
Watanabe, N., Takasaki, Y., Sato, C., Ando, S. & Tanaka, I. Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations. Acta Cryst. D 65, 1326–1333 (2009).
Article CAS Google Scholar
Blatch, G. L. & Lässle, M. The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays 21, 932–939 (1999).
Article CAS PubMed Google Scholar
Fairman-Williams, M. E., Guenther, U.-P. & Jankowsky, E. SF1 and SF2 helicases: family matters. Curr. Opin. Struct. Biol. 20, 313–324 (2010).
Article CAS PubMed PubMed Central MATH Google Scholar
Wu, B. et al. Structural basis for dsRNA recognition, filament formation, and antiviral signal activation by MDA5. Cell 152, 276–289 (2013).
Article CAS PubMed MATH Google Scholar
Wang, W. & Pyle, A. M. The RIG-I receptor adopts two different conformations for distinguishing host from viral RNA ligands. Mol. Cell 82, 4131–4144.e6 (2022).
Article CAS PubMed PubMed Central MATH Google Scholar
Kennedy, M. A. et al. Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI. Nucleic Acids Res. 51, 4467–4487 (2023).
Article CAS PubMed PubMed Central MATH Google Scholar
Teichmann, M., Dumay-Odelot, H. & Fribourg, S. Structural and functional aspects of winged-helix domains at the core of transcription initiation complexes. Transcription 3, 2–7 (2012).
Article PubMed MATH Google Scholar
Arcus, V. OB-fold domains: a snapshot of the evolution of sequence, structure and function. Curr. Opin. Struct. Biol. 12, 794–801 (2002).
Article CAS PubMed MATH Google Scholar
Theobald, D. L., Mitton-Fry, R. M. & Wuttke, D. S. Nucleic acid recognition by OB-Fold proteins. Annu. Rev. Biophys. Biomol. Struct. 32, 115–133 (2003).
Article CAS PubMed PubMed Central MATH Google Scholar
Chen, W.-F. et al. Molecular mechanistic insights into drosophila DHX36-mediated g-quadruplex unfolding: a structure-based model. Structure 26, 403–415.e4 (2018).
Article CAS PubMed MATH Google Scholar
Song, X. et al. Catalytically inactive long prokaryotic Argonaute systems employ distinct effectors to confer immunity via abortive infection. Nat. Commun. 14, 6970 (2023).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Wang, S. et al. Landscape of new nuclease-containing antiphage systems in Escherichia coli and the counterdefense roles of bacteriophage T4 genome modifications. J. Virol. 97, e00599–23 (2023).
Article PubMed PubMed Central Google Scholar
Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).
Article CAS PubMed PubMed Central MATH Google Scholar
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Article CAS PubMed Google Scholar
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Article CAS PubMed PubMed Central MATH Google Scholar
Chen, S. et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013).
Article CAS PubMed PubMed Central MATH Google Scholar
Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).
Article CAS PubMed MATH Google Scholar
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D Biol. Crystallogr. 71, 136–153 (2015).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D Struct. Biol. 75, 861–877 (2019).
Article ADS CAS PubMed PubMed Central MATH Google Scholar
Williams, C. J. et al. MolProbity: more and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Article CAS PubMed MATH Google Scholar
Barad, B. A. et al. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).
Article CAS PubMed PubMed Central MATH Google Scholar
Hothorn, M. et al. Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase. Science 324, 513–516 (2009).
Article ADS CAS PubMed MATH Google Scholar
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Article CAS PubMed PubMed Central MATH Google Scholar
Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).
Article PubMed PubMed Central Google Scholar
Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Article CAS PubMed MATH Google Scholar
Letunic, I. & Bork, P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res. 49, W293–W296 (2021).
Article CAS PubMed PubMed Central Google Scholar
Gabler, F. et al. Protein sequence analysis using the mpi bioinformatics toolkit. Curr. Protoc. Bioinform. 72, e108 (2020).
Article CAS MATH Google Scholar
Wang, J. et al. The conserved domain database in 2023. Nucleic Acids Res. 51, D384–D388 (2023).
Article ADS CAS PubMed Google Scholar

Download references

Acknowledgements

We thank the cryo-EM Facility of University of Science and Technology of China for providing cryo-EM and computation support and the Center for Protein Research and Public Laboratory of Electron Microscopy, Huazhong Agricultural University, for technical support; Drs. Yongxiang Gao and Jianbo Cao for help with cryo-EM data collection. We thank Professor Shuai Le for providing the ΦΧ174 phage and Professor Wenyuan Han for providing the M13 phage. We thank Professors Jian Yang and Fabai Wu for their discussions regarding the bioinformatics data. T.Z. was supported by funds from the Science and Technology Innovation 2030 (2022ZD0402001-04), the National Natural Science Foundation of China (32270169, 32070174), the Foundation of Hubei Hongshan Laboratory (2021hszd013), the Fundamental Research Funds for the Central Universities (2662023PY001) and the funds of the National Key Laboratory of Agricultural Microbiology (AML2024D01).

Author information

These authors contributed equally: Yongqing Cui, Zhikang Dai, Yufei Ouyang.

Authors and Affiliations

National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China
Yongqing Cui, Zhikang Dai, Yufei Ouyang, Chunyang Fu, Yanjing Wang, Xueting Chen, Kaiyue Yang, Shuyue Zheng, Wenwen Wang, Pan Tao, Zeyuan Guan & Tingting Zou

Authors

Yongqing Cui
View author publications
Search author on:PubMed Google Scholar
Zhikang Dai
View author publications
Search author on:PubMed Google Scholar
Yufei Ouyang
View author publications
Search author on:PubMed Google Scholar
Chunyang Fu
View author publications
Search author on:PubMed Google Scholar
Yanjing Wang
View author publications
Search author on:PubMed Google Scholar
Xueting Chen
View author publications
Search author on:PubMed Google Scholar
Kaiyue Yang
View author publications
Search author on:PubMed Google Scholar
Shuyue Zheng
View author publications
Search author on:PubMed Google Scholar
Wenwen Wang
View author publications
Search author on:PubMed Google Scholar
Pan Tao
View author publications
Search author on:PubMed Google Scholar
Zeyuan Guan
View author publications
Search author on:PubMed Google Scholar
Tingting Zou
View author publications
Search author on:PubMed Google Scholar

Contributions

Z.G. and T.Z. conceived the project. Y.C., Z.D., Y.O., C.F., Y.W., X.C., S.Z., W.W., and K.Y. performed the experiments. Y.C. and Z.D. prepared the cryo-EM samples and collected cryo-EM data. Z.G. determined the structures. P.T. provided phage collections. All authors analyzed the data and contributed to manuscript preparation. Y.C., Z.D., Z.G., and T.Z. wrote the manuscript.

Corresponding authors

Correspondence to Zeyuan Guan or Tingting Zou.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information (download PDF )

Description of Additional Supplementary Files (download PDF )

Supplementary Data 1 (download DOCX )

Supplementary Data 2 (download DOCX )

Supplementary Data 3 (download XLSX )

Reporting Summary (download PDF )

Transparent Peer Review file (download PDF )

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Cite this article

Cui, Y., Dai, Z., Ouyang, Y. et al. Bacterial Hachiman complex executes DNA cleavage for antiphage defense. Nat Commun 16, 2604 (2025). https://doi.org/10.1038/s41467-025-57851-1

Download citation

Received: 07 October 2024
Accepted: 04 March 2025
Published: 17 March 2025
Version of record: 17 March 2025
DOI: https://doi.org/10.1038/s41467-025-57851-1