Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx’s dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.

Introduction
Bacteria and bacteriophages participate in an ongoing coevolutionary arms race, driving the development of sophisticated strategies for phage exploitation and host manipulation[1-3]. In response, bacteria have evolved a diverse arsenal of immune defenses, collectively known as anti-phage defense systems[4-7]. These anti-phage defense systems include innate mechanisms like restriction-modification systems, as well as adaptive strategies such as CRISPR-Cas[6, 8-10]. These systems were historically considered the primary lines of bacterial defense; recent genomic and functional analyses have uncovered an extensive array of anti-phage strategies clustered within genomic “defense islands”[3, 11, 12]. These discoveries have expanded the known bacterial immune landscape to over 154 distinct defense systems, many of which remain mechanistically uncharacterized.

Among these, the PD-(D/E)XK superfamily of nucleases, defined by a conserved catalytic core and a bipartite PD-(D/E)XK motif, constitutes a major class of phage defense systems[13-17]. These nucleases typically function as single-nuclease domain enzymes that cleave nucleic acids with high specificity[15, 18]. However, their functional diversity, regulatory mechanisms, and evolutionary adaptations remain poorly under stood. Recently, Upx has been identified as a putative single-gene defense factor within the PD-(D/E)XK family[11]. While its C-terminal domain (CTD) was predicted to adopt a canonical PD-(D/E)XK fold, the functional role of its N-terminal domain (NTD) remained enigmatic due to the absence of recognizable catalytic motifs[11]. The lack of structural and biochemical characterization has further obscured its underlying molecular mechanism. Notably, phylogenetic and genomic analyses place Upx within a clade of single-protein defense systems, raising fundamental questions about how a solitary protein can mediate robust phage resistance[19].

Here, we show Upx as an example of a single-polypeptide anti-phage system that integrates two distinct nuclease domains within a unified architecture. Using Cryo-EM, we resolve the Upx structure and reveal a tripartite organization comprising an auto-inhibited N-terminal nuclease (NTD), a central regulatory middle domain (MD), and a C-terminal PD-(D/E)XK nuclease domain (CTD)[20]. Biochemical characterization demonstrates that the CTD functions as a magnesium-dependent, nonspecific 3′–5′ exonuclease targeting both ssDNA and ssRNA, while the NTD—previously unrecognized as a nuclease—harbors a minimal PD-(D/E)XK-like catalytic core with specificity for ssDNA degradation. Notably, the MD acts as a regulatory hub, suppressing NTD activity while enhancing CTD efficiency. Our IP-MS analysis revealed that the phage protein gp16 interacts with Upx and functionally relieves the MD-mediated autoinhibition of the NTD. The phylogenetic restriction of gp16 to ΦV-1 and a narrow set of related phages provides the molecular basis for Upx’s narrow antiphage spectrum. Substrate profiling further defines Upx’s physiological preferences, providing insights into its potential in vivo targets and the functional relevance of its dual nuclease activities. Functional assays confirm that both nuclease domains cooperate to disrupt phage replication, thereby conferring resistance against bacteriophage ФV-1.Our findings redefine the functional landscape of PD-(D/E)XK nucleases by demonstrating that single-protein defense systems can evolve dual enzymatic activities through domain fusion and regulatory specialization. This work expands the mechanistic repertoire of bacterial immunity. It provides a framework for understanding how modular domain architectures drive the evolution of sophisticated anti-phage strategies within compact genetic systems.

Results
Phylogenetic analysis of Upx as a member of PD-(D/E)XK superfamily in bacterial defense system
The PD-(D/E)XK superfamily is defined by a conserved structural core known as the restriction endonuclease-like fold[13, 14]. Among the 154 prokaryotic defense systems included in the DefenseFinder Wiki (https://defensefinder.mdmlab.fr/wiki/), 13 defense systems are part of this superfamily. Notably, 8 of these, including Upx, are made up of a single gene, suggesting that they may function as solitary nuclease proteins and directly target nucleic acids in their anti-phage defense activity.

To explore the functional diversity of PD-(D/E)XK superfamily members in bacterial immunity, we aligned 1,000 representative protein sequences using MAFFT. The phylogenetic tree was generated using the IQ-TREE server and then visualized with FigTree. The analysis indicated that the 13 defense systems in PD-(D/E)XK superfamily collected in DefenseFinder span in seven clades based on sequence similarity, with Upx closely related to AbiU-1 (Fig. 1a). Previous studies have shown that AbiU-1 inhibits phage DNA transcription in infected bacterial cells[16], suggesting that Upx may confer phage resistance through a similar mechanism.

Upx protects bacterial cells from bacteriophage infections
To evaluate the phage defense capability of Upx, we challenge cells expressing Upx in plaque assays with E. coli phages representing seven distinct genera (Fig. 1b). Upx gene (NCTC6026_01102, protein SQI81149.1) from Salmonella enterica NCTC6026 was cloned into the pBAD LIC cloning vector (8A) and transformed into E. coli MG1655. Solutions comprising seven distinct bacteriophages were prepared and serially diluted 10-fold in LB medium. An aliquot of 2.5 µL from each dilution was applied onto bi-layer agar plates that had been pre-inoculated with E. coli MG1655. The assays were performed in triplicates, with the following groups: blank control (empty vector), negative control (Upx inactivating mutant E1176A/K1178A), and experimental (Upx), and the representative results are shown in Fig. 1b.

The results indicated that Upx expression conferred significant resistance to phage ФV-1 infection in E. coli MG1655 but did not provide substantial protection against the other six types of bacteriophages. However, the precise molecular mechanisms underlying this resistance remain unclear and require further investigation.

The Cryo-EM structure of Upx
To elucidate the molecular mechanisms underlying the anti-phage capability of Upx, we expressed and purified the Upx protein from Escherichia coli BL21(DE3). Results of the Gel filtration chromatography experiment using a Superdex 200 column showed an eluting peak at 14.3 mL, suggesting that Upx likely exists as a monomer in solution (Fig. 1c), a conclusion further supported by analytical ultracentrifugation (AUC) experiments (Supplementary Fig. 1a).The result of the SDS-PAGE assay showed that the molecular weight of Upx is approximately 143 kDa, with sufficient purity for subsequent experiments (Fig. 1d).

The structural characteristics of Upx were analyzed using cryo-EM, resulting in a structure with a high resolution of 3.01 Å (Supplementary Fig. 1b-d, Supplementary Fig. 2; Supplementary Table 1). The cryo-EM structure confirmed that Upx adopts a symmetric monomeric conformation, consistent with the Gel filtration chromatography and AUC results (Fig. 2b, c; Supplementary Fig. 1a). A DALI search against the PDB database yielded no significant structural homologs, indicating that Upx adopts a fold that is distinct from those of previously characterized proteins (Supplementary Fig. 3). Upx consists of three distinct domains: the N-terminal domain (NTD, residues 1–515), the middle domain (MD, residues 516–714), and the C-terminal domain (CTD, residues 715–1272), arranged in a fusiform shape with the NTD and CTD at opposite ends and the MD in the center (Fig. 2a-c). Although the three domains are relatively distinct, they are structurally interconnected and likely function cooperatively.

Upx is a magnesium-dependent 3'-5' exonuclease targeting ssDNA
Having resolved the structure of Upx, we next sought to characterize its nuclease activity. To this end, we performed cleavage assays using single-stranded DNA (ssDNA) and RNA (ssRNA) substrates labeled with Cy3 fluorophores at either the 5’ or 3’ ends. Upx efficiently degraded 5’ Cy3-labeled ssDNA into progressively shorter fragments, producing a degradation pattern characteristic of exonuclease activity (Fig. 3a). In contrast, no cleavage was detected for the 3’ Cy3-labeled ssDNA substrate (Supplementary Fig. 4a), suggesting that the bulky Cy3 moiety at the 3’ terminus prevented enzymatic access and thereby revealed the 3′-5′ directionality of nuclease activity. Similarly, while Upx only weakly acted upon 5’ Cy3-labeled ssRNA, it showed no activity 3’-labeled ssRNA, further supporting its role as a 3′-5′ exonuclease (Fig. 3b; Supplementary Fig. 4b). To validate this observation, we introduced three phosphorothioate linkages at the 3’ end of ssDNA, a modification known to block exonuclease cleavage. As anticipated, Upx completely lost nuclease activity against this modified substrate (Fig. 3c). By contrast, no detectable cleavage was observed on double-stranded DNA (dsDNA), indicating that Upx selectively targets single-stranded nucleic acids (Fig. 3d).

We next investigated whether Upx exhibits sequence or length selectivity. Substrates of different lengths (60 nt, 50 nt, and 30 nt) and different sequences were all efficiently degraded, indicating that Upx functions as a sequence- and length-independent exonuclease (Fig. 3e).Similarly, ssRNA substrates of varying lengths (45 nt and 60 nt) were also cleaved, albeit with markedly lower efficiency compared to ssDNA (Fig. 3b; Supplementary Fig. 4c).Finally, we examined the requirement for divalent metal ions. To explore the metal ion dependency of Upx’s nuclease activity, we examined seven divalent metal ions: magnesium (Mg²⁺), iron (Fe²⁺), nickel (Ni²⁺), cerium (Ce²⁺), cobalt (Co²⁺), zinc (Zn²⁺), and Manganese (Mn2+). The findings revealed that the degradation of ssDNA by Upx and CTD was facilitated by magnesium (Mg²+). Manganese (Mn2+) also supported substantial activity, while iron (Fe²⁺) permitted only trace cleavage. The remaining cations did not support significant activity (Fig. 3f; Supplementary Fig. 5a).

The CTD harbors a canonical PD-(D/E)XK catalytic core
Given that Upx is composed of three domains (NTD, MD, and CTD), we next sought to determine which domain is responsible for its nuclease activity. To this end, we purified truncation constructs corresponding to individual domains (NTD, CTD) and paired domains (NTD–MD, MD–CTD) and evaluated their nuclease activity. The MD–CTD truncation exhibited cleavage activity comparable to that of full-length Upx, whereas CTD alone retained partial activity on both ssDNA, but not dsDNA (Fig. 3d, g). Interestingly, the NTD construct displayed detectable nuclease activity, but this activity was abolished when fused with the MD (Fig. 3g-h), suggesting that MD may exert a regulatory effect on NTD-mediated catalysis, which we investigated in detail in a later section.

The PD-(D/E)XK family members share a common structural core, comprising four or five β-sheets flanked on both sides by α-helices. This core is often embedded within highly variable peripheral regions. The central β-sheet serves as a scaffold for a weakly conserved active site, typically comprising two or three acidic residues (Asp or Glu) along with a Lys residue, collectively forming the characteristic bipartite catalytic motif PD-(D/E)XK, where X is any amino acid[21, 22].To definitively classify the CTD, we performed a structural alignment with the DALI server, which identified it as a canonical member of the PD-(D/E)XK superfamily (Fig. 3i; Supplementary Fig. 4d-i). The core domain (residues 1121–1272) forms the characteristic fold of two α-helices and four β-sheets, featuring a sharply kinked α2 helix that, together with α1, defines the active site pocket (Fig. 3i). The conserved active site is defined by key residues D1161 in β2 and E1176/K1178 in β3, resembling the active site architecture of the restriction enzyme FokI and other members of the PD-(D/E)XK superfamily (Supplementary Fig. 4d-i). This structural evidence further confirms that Upx is a representative member of the PD-(D/E)XK superfamily from a structural biology perspective.

To explore the metal ion dependency of CTD’s nuclease activity, we examined six divalent metal ions: magnesium (Mg²⁺), iron (Fe²⁺), nickel (Ni²⁺), cerium (Ce²⁺), cobalt (Co²⁺), and zinc (Zn²⁺). The findings revealed that the degradation of ssDNA by CTD was facilitated by magnesium (Mg²+) and, to a lesser extent, iron (Fe²+). At the same time, the other tested divalent cations did not significantly affect cleavage activity (Fig. 3j).

The NTD contains an atypical simplified PD-(D/E)XK catalytic core
Cryo-EM analysis revealed that the NTD harbors a distinct, negatively charged pocket consistent with a potential nuclease active site (Fig. 4a, b). Biochemical assays confirmed that the NTD functions as a magnesium-dependent 3′–5′ exonuclease selectively targeting ssDNA, whereas no cleavage was detected toward ssRNA or dsDNA substrates (Fig. 4c; Supplementary Fig. 4c). Structure-guided mutagenesis identified five charged residues—D122, D127, R129, E147, and D173—as critical for catalysis, since their substitution markedly impaired or abolished nuclease activity (Fig. 4d; Supplementary Fig. 5b-e). Structural inspection showed that D127 and R129 form a DXR motif on β2, analogous to the canonical DXK catalytic triad of PD-(D/E)XK nucleases, while D122, E147, and D173 reside on β1, α1, and α2, respectively (Fig. 4b, g). Unlike the classical PD-(D/E)XK fold comprising four or five β-strands, the NTD active site is formed by only two β-strands and two flanking helices, representing a simplified yet catalytically competent variant of this nuclease scaffold. Domain topology comparison further revealed that while the Upx CTD adopts the canonical PD-(D/E)XK architecture, the NTD constitutes a noncanonical, minimalist version, serving as a structurally convergent counterpart (Fig. 4e-g). Collectively, these findings identify a previously unrecognized nuclease topology, expanding the structural and functional diversity of the PD-(D/E)XK superfamily.

The MD structurally integrates and functionally regulates the NTD and CTD
The three domains of Upx form an integrated structural unit, with the middle domain (MD) serving as a central hub that physically connects the NTD and CTD (Fig. 5a). This architecture provides the structural basis for its regulatory role. Detailed analysis shows distinct interaction patterns at the two interfaces: the MD–NTD interface is dominated by electrostatic interactions involving residue pairs D122–R517, R183–E562, R183–D514, and D514–R530 (Fig. 5b), while the MD–CTD interface employs a more diverse stabilization strategy through hydrogen bonds (R595–L820, N705–G819, R707–L818), electrostatic interactions (R541–E824), and π–π stacking (F536–W810) (Fig. 5c). Functionally, the MD exerts differential regulation on the two nuclease domains. It enhances CTD activity—while the isolated CTD exhibits only weak cleavage activity, fusion with the MD (MD–CTD) significantly improves nuclease efficiency on ssDNA (Fig. 3g). This enhancement is corroborated by EMSA, where CTD alone fails to produce a detectable shift with ssDNA, likely due to transient binding, while the MD–CTD construct yields a clear shift, indicating that the MD promotes stable substrate engagement (Fig. 5d).

Conversely, the MD inhibits the activity of NTD. Although the isolated NTD cleaves ssDNA efficiently, fusion with the MD (NTD–MD) abolishes this activity (Fig. 3g). Structural analysis shows that the MD partially occludes the NTD catalytic pocket (Fig. 5a), restricting substrate access. Consistently, EMSA revealed that the isolated NTD binds ssDNA effectively, whereas the NTD–MD construct shows no mobility shift, confirming that the MD suppresses both substrate binding and cleavage by the NTD (Fig. 5d).

To explore the physiological regulation of Upx, we performed MBP-tagged IP–MS under phage infection conditions (Supplementary Fig. 6a). This identified several phage-derived interactors, and pull-down assays confirmed direct binding between Upx and the phage protein gp16 as well as DNA polymerase (Fig. 5e; Supplementary Fig.6b; Supplementary Table 2). Functional assays showed that gp16—a large multimeric virion structural protein that forms part of the phage injection apparatus and engages the host during the earliest stage of infection—specifically relieves MD-mediated inhibition and restores NTD activity in both the NTD-MD fragment and full-length Upx, whereas DNA polymerase showed no such activating effect (Fig. 5f; Supplementary Fig.6c,f). Pull-down mapping revealed that gp16 binds broadly to multiple Upx domains (NTD, CTD, NTD–MD, and MD–CTD), but not to the MBP control (Supplementary Fig. 6d, e), suggesting a potential activation mechanism in which gp16 counteracts MD repression to unlock NTD nuclease function during infection.Phylogenetic analysis shows that gp16 is uniquely present in ΦV-1 and a narrow set of related phages, but absent in all Upx-insensitive phages tested, including T6, Mu, P1, SP, T1, and vB (Supplementary Fig. 7; Supplementary Fig.8). This restricted distribution provides a mechanistic explanation for the narrow antiphage spectrum of Upx, linking its activation to a phage-encoded specificity determinant.

Substrate profiling reveals Upx's preference for phage replication intermediates
Since ΦV-1 is a dsDNA phage yet Upx lacks dsDNA cleavage activity, its physiological substrates are likely single-stranded nucleic acid intermediates generated during essential processes such as replication, transcription, or recombination. To delineate the substrate selectivity of Upx, we examined its activity toward a panel of structured nucleic acids mimicking physiological intermediates, including 5′ overhangs, 3′ overhangs, fork structures, D-loops, and R-loops (Fig. 5g-m; Supplementary Table 3, 4). Full-length Upx preferentially cleaved substrates bearing unpaired 3′ ssDNA termini, such as 3′ overhangs and fork structures (Fig. 5g, j, k), but showed no activity against 5′ overhangs or 3′ ends blocked with a Cy3 moiety. Given that DNA-RNA hybrids and D-loop intermediates frequently arise during phage transcription, replication, and host recombination, we next asked whether Upx could process these complex substrates. Notably, Upx exhibited robust activity against D-loop structures but only trace activity against R-loops (Fig. 5l). To further dissect its cleavage specificity, we designed additional D-loop variants and found that Upx only cleaved those possessing a 3′ overhang (Fig. 5m), consistent with its identified 3′-5′ exonuclease activity. In parallel, IP-MS suggested a possible interaction between Upx and phage DNA polymerase, which was subsequently confirmed by pull-down assays (Fig. 5e).

Collectively, these results support a model in which Upx targets single-stranded nucleic acid intermediates—such as those generated during phage genome replication, transcription, or host recombination—to exert anti-phage activity.

Upx suppresses phage genome accumulation and reprograms host transcription during infection
To directly assess the impact of Upx on phage genome dynamics during infection, we performed whole-genome sequencing at early (5 min) and late (20 min) time points following phage challenge in E. coli strains expressing wild-type Upx, a catalytically inactive Upx mutant (Upx mutation), or an empty vector control. In both intervals, normalized relative DNA abundance of phage genomes was markedly reduced in the Upx-expressing cells compared with either the vector control or the nuclease-inactive mutant (Fig. 6a), demonstrating that Upx efficiently cleaves incoming phage DNA immediately upon infection and continues to suppress its accumulation through later stages.

To determine how Upx interferes with the transcriptional progression of ΦV-1 infection, we performed RNA-seq on infected cells with or without Upx expression. Hierarchical clustering revealed a strong separation between the two groups (Fig. 6b), indicating that Upx profoundly reshapes the transcriptional landscape during phage attack. Direct analysis of phage-derived transcripts showed that every detectable ΦV-1 gene was markedly downregulated in the presence of Upx (Fig. 6c), demonstrating that Upx effectively suppresses phage transcription after infection. This result is fully consistent with Upx-mediated degradation of incoming phage genomes, which prevents the initiation of viral gene expression. GO analysis of the downregulated genes further revealed that Upx blocks multiple cellular processes required for phage propagation (Fig. 6d). Pathways associated with the viral life cycle—including “viral process”, “viral life cycle”, “virus tail”, and other virion structural components—were among the most strongly suppressed. In addition, Upx reduced expression of structural and motility pathways such as bacterial-type flagellum assembly, cell projection organization, and periplasmic or membrane-associated components, all of which are known to be extensively remodeled during productive phage infection.

Together, these results show that Upx imposes a phage-restrictive state at both the DNA and RNA levels. Upx prevents effective amplification of the phage genome, leading to a marked reduction in viral DNA accumulation. In the meantime, Upx imposes a phage-restrictive transcriptional state: by destroying phage DNA, Upx eliminates viral transcripts at their source and prevents the activation of both viral gene expression programs and the host pathways that ΦV-1 normally exploits during its replication cycle.

Anti-phage function requires cooperation of NTD and CTD
To directly assess the contribution of each domain to phage defense, we compared the anti-phage activity of full-length Upx with truncation and point-mutant variants. Deletion of either the NTD or the CTD completely abolished phage resistance, demonstrating that both nuclease domains are indispensable for defensive function (Fig. 7a). Consistently, expression of individual domains or partial constructs failed to confer measurable protection, underscoring that the cooperative action of all domains is required (Fig. 7a). Furthermore, alanine substitutions of residues critical for catalysis within either the NTD (D127A/R129A) or the CTD (E1176A/K1178A) also eliminated phage resistance, confirming that the enzymatic activities of both domains are essential in vivo (Fig. 7a). We also introduced alanine substitutions at residues located at the MD–NTD or MD–CTD interfaces (e.g., R530A/E562A/R517A or R707A/R595A/R541A/N705A). Interestingly, none of these mutations impaired the anti-phage activity of Upx, highlighting an unexpected layer of complexity in its regulation (Fig. 7b). These findings establish that Upx-mediated defense relies on the coordinated contributions of the NTD and CTD, integrated through MD-dependent regulation. To elucidate the system-level defense strategy of Upx, we investigated whether it functions via an abortive-infection or direct immunity mechanism. Following the classification criteria described previously[12], we performed phage-infection assays across a range of multiplicities of infection (MOIs). In E. coli strains overexpressing Upx, rapid bacterial growth was observed consistently at both low (MOI=0.1) and high (MOI=3) infection multiplicities (Fig. 7c). This growth pattern, where robust proliferation occurs regardless of phage dose, demonstrates that Upx provides direct immunity to infected cells rather than employing an abortive-infection strategy that sacrifices individual cells. In conclusion, Upx represents a previously uncharacterized dual-nuclease domain defense protein, functioning as a magnesium-dependent, non-sequence-specific 3′-5′ exonuclease active on ssDNA and ssRNA. The NTD emerges as a previously uncharacterized nuclease domain whose activity is autoinhibited by the MD, while the CTD displays robust nuclease activity that is potentiated by the MD. The ability of phage protein gp16 to relieve MD-mediated inhibition of the NTD highlights a dynamic regulatory mechanism that may be engaged during infection. Collectively, our study defines Upx as a previously unrecognized single-protein dual-nuclease defense system, expanding the functional and mechanistic repertoire of bacterial immunity (Fig. S7d).

Methods
Phylogenetic analysis of PD-D (E)XK nuclease
The phylogenetic analysis of the PD-(D/E)XK superfamily was performed utilizing a dataset comprising 83,737 protein sequences obtained from UniProt. In order to improve data integrity and minimize redundancy, Proteins of eukaryotic origin or those with fewer than 100 amino acids were excluded from the analysis, and then de-duplication was carried out to eliminate sequences with more than 70% similarity. This process resulted in a final dataset consisting of 1,000 representative sequences. Multiple sequence alignment was performed using the MAFFT (version 7)[25, 26], while phylogenetic reconstruction was executed using the IQ-TREE (v3.0.1)[27], which incorporated standard model selection. The generated phylogenetic tree was visualized using FigTree software (v1.4.4).

Plasmid construction
The genes of interest were amplified utilizing PrimeSTAR® HS DNA Polymerase (Takara), employing primers that possess 5' ends complementary to the ends of the plasmid backbone. The linear vector backbone was generated through PCR amplification or restriction enzyme digestion at multiple cloning site (MCS) locations. The assembly of plasmids was conducted using the In-Fusion® HD Cloning Kit (Clontech). Upx and mutant constructs were assembled into the pLIB vector respectively, with an N-terminal TEV cleavable His-ZZ-tag, for downstream purification via ZZ-tag affinity. Genes inserted into pLIB vector were checked via Sanger sequencing (Source Bioscience, Cambridge) before downstream steps.

Protein expression and purification
The genes of interest were successfully cloned into the pET-28-MKH8SUMO vector, which features an N-terminal 8×His-SUMO tag. The resultant plasmids were transformed into E. coli BL21 (DE3) cells to facilitate protein expression in LB medium. Protein expression was achieved by adding 0.2 mM IPTG at 16 °C overnight. After the protein expression induction period, the cells were harvested and then lysed via sonication in binding buffer (25 mM Tris–HCl, 500 mM NaCl, 5% glycerol, 5 mM imidazole, and, 2 mM β‐mercaptoethanol, pH 8.0) on ice. After centrifugation, the supernatants were incubated with the Ni-NTA resin (Qiagen) for 1 h and subsequently washed thoroughly with washing buffer (25 mM Tris–HCl, 500 mM NaCl, 5% glycerol, 15 mM imidazole, and 2 mM β‐mercaptoethanol, pH 8.0). The His-tagged target proteins were then eluted using an elution buffer (25 mM Tris–HCl, 300 mM imidazole, 500 mM NaCl, 5% glycerol, and 2 mM β‐mercaptoethanol, at pH 8.0). The eluted samples were subsequently applied to a heparin column (GE Healthcare) and eluted using a linear NaCl gradient, followed by size-exclusion chromatography utilizing a Superdex 200 column (GE Healthcare), the buffer consisting of 25 mM Tris–HCl, 2 mM DTT, 150 mM NaCl, at pH 8.0. Protein peak fractions were collected and concentrated, the protein concentration were determined for subsequent experiments.

The N-terminal His-SUMO tag of Upx and truncation mutants protein can be cleaved by adding the TEV protease at the molar ratio of 1:10, and the tag can be removed by re-incubating with Ni-NTA resin. The resulting tag-free Upx and truncation mutant protein can then undergo further purified using HiTrap Heparin HP column (Cytiva) then Superdex 200 column (Cytiva). The protein samples were subsequently analyzed using 10% SDS-PAGE. The purified protein samples that are not immediately utilized would be stored at −80 °C.

Plaque assays and bacteriophage cultivation
The bacteriophages vB, Mu, SP, T6, T1, P1, and ФV-1, along with the host E. coli strain MG1655 (ATCC 25404), were ordered from the American Type Culture Collection (ATCC). The E. coli MG1655 strain was cultured in LB medium until OD600 reached 0.4. Subsequently, the bacteriophage was introduced into the host strain in a 1:2 ratio and incubated for 5 to 8 h. Upon observing a transition of the culture medium from turbidity to clarity. The lysate was subjected to centrifugation at 4696g for 15 min to eliminate cellular debris. Subsequently, the resultant supernatant was filtered using a 0.22 µm filter.

The phage titer was assessed by the small-drop plaque assay method [28]. For the plaque assays, freshly cultured E. coli MG1655 (OD₆₀₀ = 0.8) transformed with different constructs was mixed with semi-solid medium containing 50 mg/L ampicillin and 200 mg/L arabinose at a 1:100 (v/v) ratio. This mixture was then poured onto a 15 cm LB agar plate and allowed to stand for 1 h to become solid. Phage solutions ranging from 106 to 1011 PFU/mL were diluted with LB in a 10-fold series and then dropped onto a bilayer solid agar plate at 2.5 uL per drop and repeated in triplicate. The plates were allowed to dry for 30 min before being incubated for 12 h at 37 °C.

In vitro DNA/RNA cleavage assays
The DNA/RNA substrates were incubated with purified Upx at 37 °C for 30 min in a 20 μ L DNA cleavage reaction buffer containing 25 mM Tris at pH 8.0, 2 mM DTT, 150 mM KCl, 2 mM MgCl2, and 5% Glycerol unless otherwise stated. After incubation, reactions were stopped with DNA loading buffer containing 60 mM EDTA and 5 μL was analyzed on a 13% Urea-PAGE gel, which was run at 240 V for 5 min followed by 150V for 1 h. The gel was then analyzed on ChemiDoc MP Imaging System. To test activity in the presence of different divalent cations (Mg²⁺, Mn²⁺, Fe²⁺, Zn²⁺, Ni²⁺, Co²⁺, or Ce²⁺), Upx (or its truncations) was treated with 10 mM EDTA on ice for 30 min and then exchanged into metal‑free buffer using a desalting column. Cleavage reactions were carried out as described above, except that the reaction buffer was replaced with 20 mM Tris-HCl (pH 8.0) and 50 mM NaCl, supplemented with 0.2 mM of the indicated metal chloride or sulfate salt.

Cryo-EM sample preparation and data collection
For the Upx protein sample, 4 μL of 1 mg/mL were applied to a glow-discharged Quantifoil holey carbon grid (Au, R1.2/1.3, 300 mesh). The grids were subjected to blotting with a force of 2 for 2 seconds, after which they were rapidly plunged into liquid ethane using a Vitrobot Mark IV (Thermo Scientific). Cryo-EM grids were screened using a Talos Arctica microscope (ThermoScientific) operating at 200 kV, and the final map was collected on a Titan Krios G4 (FEI) operating at 300 kV. Images were captured through the Smart EPU software (v2.8.1) at a nominal magnification of 165, 000×, resulting in a calibrated physical pixel size of 0.692 Å/pixel, with a defocus range from -1.2 μm to -2.2 μm. Imaging was performed in super-resolution mode on a K3 summit electron direct detector, positioned at the output of a GIF-Quantum energy filter set to a slit width of 20 eV. Data were collected at a frame rate of 32 frames per second. The total electron dose was 50 e-/Å2. In total, 6,685 movie stacks were collected.

Cryo-EM image processing
The movie frames were processed using cryoSPARC (v4.7.1)[29], aligned with MotionCor2 (v1.6.4)[30] at a binning factor of 2, and underwent real-time contrast transfer function (CTF) estimation using Gctf (v1.06)[31]. A total of 1,025,867 particles were automatically identified using the topaz picking (v0.2.5)[22], and 615,173 particles were retained for further analysis after 2D classification to eliminate poor-quality particles. Following the ab-initio reconstruction, a final set of 204, 860 particles were selected for non-uniform (NU) refinement, resulting in a resolution of 3.01 Å.

Model building and refinement
The atomic model of Upx, as predicted by AlphaFold3, was utilized as our initial template. We proceeded to segment this preliminary model according to its domains and subsequently docked these segments into the cryo-electron microscopy (cryo-EM) map using ChimeraX (v1.9)[32, 33]. The comprehensive model was then subjected to manual refinement in Coot (v0.9.7)[34]. Following this, we performed iterative real-space refinements utilizing Phenix.real_space_refine (v1.20.1-4487)[35]. To evaluate the quality of the model, we utilizedMolProbity within the Phenix suite (v1.20.1-4487)[36], and detailed refinement statistics are available in Table S1.

Electrophoretic mobility shift assay (EMSA)
The EMSA was conducted in a cleavage buffer with the addition of 10 mM EDTA, and incubated at 37°C for 1 h. Purified Upx or different protein constructs were mixed with ssDNA in a 40:1 molar ratio. The ssDNA sequences utilized for the EMSA were nonspecific and labeled with 5' Cy3. The reaction mixture was examined using a 6% native PAGE gel, which was run for 1 h at 120 V at 4 °C. The fluorescence signal was captured with the Tanon 5200 imaging system.

Co-immunoprecipitation coupled with mass spectrometry (Co-IP–MS)
There were three experimental groups: MBP (Blank control), MBP + ΦV-1 infected (control), and MBP–Upx + ΦV-1 infection, with two biological replicates per group. E. coli MG1655 cells overexpressing MBP or MBP–Upx were grown to an OD of 0.3and either infected with phage ΦV-1 or left uninfected as indicated. Cells were cultured for an additional 20 min following infection, harvested by centrifugation and lysed in Co-IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA), supplemented with protease inhibitors. The lysates were incubated with MBP beads for 1 h at 4 °C, followed by extensive washing to remove nonspecific proteins. Beads were washed twice with 50 mM NH₄HCO₃ and proteins were reduced with 5 mM dithiothreitol at 56 °C for 1 h, alkylated with 15 mM iodoacetamide at room temperature for 45 min in the dark, and quenched with 30 mM cysteine. Proteins were digested on beads with trypsin (Promega) overnight at 37 °C. Peptides were collected, dried, and desalted using μ-C18 ZipTips (Millipore).

Peptides were resuspended in 0.1% formic acid and analyzed by nanoLC–MS/MS using an EASY-nLC 1200 system coupled to an Orbitrap Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were separated on a C18 column at 300 nL/min using a linear acetonitrile gradient. MS data were acquired in data-dependent mode with a Top15 method. Full MS scans were acquired at a resolution of 70,000 (m/z 350–1750), and MS/MS scans at a resolution of 17,500.

Raw data were processed with Proteome Discoverer (v3.0) and searched against the UniProtKB/Swiss-Prot phage ΦV-1proteome (November 6, 2023). Trypsin specificity with up to two missed cleavages was used. Carbamidomethylation of cysteine was set as a fixed modification, and methionine oxidation and N-terminal acetylation as variable modifications. Protein identifications were filtered at an FDR ≤0.01. Label-free quantification was performed using MS1 intensities, requiring at least two peptides per protein. Proteins with a fold change >2 and p <0.01 were considered significantly altered. Three biological replicates were analyzed per condition.

Sample preparation for RNA sequencing
E. coli MG1655 cultures were grown in LB at 37 °C with shaking (200 rpm) to an OD₆₀₀ of 0.5 and subsequently infected with Escherichia phage phiV-1 at an MOI of 3. After infected for 20 minutes, culture samples were rapidly quenched and collected by centrifugation at 4 °C, and stored at −80 °C. Total RNA was isolated using the TRIzol methods, including chloroform phase separation, isopropanol precipitation, ethanol washing, and TURBO DNase treatment. RNA quality (RIN>6.5) was identified prior to bacterial rRNA depletion and preparation of strand-specific sequencing libraries. Libraries were sequenced on an Illumina platform, and trimmed reads were aligned to a combined E. coli MG165 5 + phiV-1 reference genome for gene-level quantification and differential expression analysis.

Bioinformatic analysis for RNA-seq data
Differential expression analysis used the DESeq2 Bioconductor package (v1.34.0), a model based on the negative binomial distribution. After adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate, Padj of genes were setted <0.05 to detect differential expressed ones. Significantly differentially expressed genes (DEGs) were defined as if the genes met the criteria of Padj < 0.05 and absolute log2 fold change (|log2FC|) > 1.

The genome sequences of Escherichia phage phiV-1 were aligned against the Swiss-Prot database, and the corresponding GO terms were then obtained based on the mapping relationship between gene IDs and GO IDs. We merged the gene ID-to-GO ID mappings of Escherichia coli and Escherichia phage phiV-1 to facilitate the subsequent construction of a customized OrgDb package. AnnotationForge R package (v1.36.0) was used building customized SQLite-based annotation OrgDb package. ClusterProfiler (v4.2.2) R package was used identifying Gene Ontology (GO) terms that annotate a list of significantly DEGs. Significantly enriched terms were filtered to retain those with q-value < 0.05.

Sample Preparation for DNA-seq data (WGS data)
E. coli MG1655 cultures harboring the indicated plasmids were grown in LB medium supplemented with 0.2% (w/v) L‑arabinose to an OD of 0.5 and then infected with Escherichia phage ΦV‑1 at a MOI of 3. After 5 or 20 min of infection, cultures were rapidly chilled on ice, harvested by centrifugation at 4 °C, and stored at ‑80 °C. Total genomic DNA was extracted using the Bacterial Genomic DNA Extraction Kit (DP302, TIANGEN). DNA concentration and purity were measured with a NanoDrop 2000 spectrophotometer.

Library Construction and Sequencing
For DNA sequencing, genomic DNA (200 ng) was fragmented to ~300–350 bp using a Covaris ultrasonicator. Fragmented DNA was end‑repaired, 5′‑phosphorylated, 3′‑A‑tailed, and ligated to sequencing adaptors. Libraries were size‑selected using DNA cleanup beads and amplified by PCR for eight cycles. Library quality was assessed on an Agilent 2100 Bioanalyzer. Qualified libraries were sequenced in paired‑end mode (PE150) on Illumina platforms (Novaseq6000).

Bioinformatic Processing
Raw sequencing reads were processed with fastp (v0.23.0) to remove adaptor sequences, PCR primers, reads containing >14 ambiguous bases (N), and low‑quality reads with >40% of bases below Q20. Clean reads were aligned to a combined reference genome consisting of the E. coli MG1655 chromosome (GenBank accession: U00096.3) and the complete phage ΦV‑1 genome (NC_001426.1) using the Sentieon pipeline (v202112.02). Duplicate reads were removed, and single‑nucleotide variants (SNVs) and insertions/deletions (InDels) were called using Sentieon’s variant‑calling modules.

Read‑Depth (RD) Analysis and Normalization
To determine the relative abundance of phage DNA, we performed read‑depth (RD) analysis following principles established for copy‑number variation (CNV) detection [37]. The analysis was carried out with CNVnator (v0.3.1) using a bin size of 100 bp. A sample‑specific genomic baseline was established by analyzing the global distribution of RD across the entire combined reference genome. The RD for each genomic segment was then normalized against this internal baseline. Phage genome abundance was quantified as the mean normalized RD across all bins spanning the combined reference genome. This value represents the relative copy number of phage DNA per host‑genome equivalent, where a value of 1.0 corresponds to one phage genome per bacterial genome. The normalized RD values reflect the relative ratio of phage‑to‑host DNA in each sample.

Growth curve assays
Overnight bacterial cultures were diluted 1:100 in LB and grown to an OD₆₀₀ of 0.3. Subsequently, 180 μl of each culture was dispensed into a 96-well plate preloaded with 20 μl of either LB (uninfected control) or phage lysate to achieve final MOIs of 3 or 0.1. Plates were incubated at 25°C with shaking in microplate reader, and OD₆₀₀ readings were recorded every 15 min. Infections were set up in three independent biological replicates. Three wells containing only medium served as blanks, and their readings were subtracted from all sample OD measurements.

Data availability
The atomic coordinates data generated in this study have been deposited in the Protein Data Bank under accession code 9WUF [http://dx.doi.org/10.2210/pdb9WUF/pdb], and the electron microscopy map has been deposited in the Electron Microscopy Data Bank under accession code EMD-66260 [https://www.ebi.ac.uk/emdb/EMD-66260]. The RNA sequencing data has been deposited to Gene Expression Omnibus (GEO) database under accession code GSE311613 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE311613] (RNA-seq profiling of E. coli and their overexpression derivatives (UPX overexpressed) infected with Escherichia phage ΦV-1.Samples were collected after infected for 20 minutes.).The whole-genome resequencing data of E. coli and phage samples generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database. Corresponding BioSample accessions are SAMN53654015 [https://www.ncbi.nlm.nih.gov/sra/?term=SAMN53654015] (Control groups at 5 min after ΦV-1 infection), SAMN53654016 [https://www.ncbi.nlm.nih.gov/sra/?term=SAMN53654016] (Control groups at 20 min after ΦV-1 infection), SAMN53654017 [https://www.ncbi.nlm.nih.gov/sra/?term=SAMN53654017] (UPXS mutation groups at 5 min after ΦV-1 infection), SAMN53654018[https://www.ncbi.nlm.nih.gov/sra/?term=SAMN53654018] (UPX mutation groups at 20 min after ΦV-1 infection), SAMN53654019 [https://www.ncbi.nlm.nih.gov/sra/?term=SAMN53654019] (UPX wild-type groups at 5 min after ΦV-1 infection), and SAMN53654020 [https://www.ncbi.nlm.nih.gov/sra/?term=SAMN53 654020] (UPX wild-type groups at 20 min after ΦV-1 infection). The sequence data submissions are linked to these BioSample accessions, and all data can be accessed via the NCBI SRA portal (https://www.ncbi.nlm.nih.gov/sra) or the associated BioProject page (accession number: PRJNA1370017 [https://www.ncbi.nlm.nih.gov/bioproject/ PRJNA1370017] ). Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The discovery and characterization of bacterial defense systems are under extensive investigation. In this study, Zhou, Wei, and colleagues characterized the Upx nuclease, revealing a unique dual-nuclease architecture. As shown by biochemical and structural analysis, Upx contains two nuclease active sites: a classical C-terminal PD-(D/E)XK nuclease (CTD) and an atypical N-terminal PD-(D/E)XK nuclease (NTD). Functional analyses show that the CTD can cleave both ssDNA and ssRNA, whereas the NTD is specific for ssDNA. The authors further reported the cryo-EM structure of apo Upx, providing structural insights into the active site features of both NTD and CTD. Additionally, Upx harbors a middle domain (MD) that differentially regulates its nuclease activities—stimulating CTD while inhibiting NTD. While these findings are intriguing, additional experiments are required to fully elucidate the mechanism of Upx function\n1. The authors demonstrate that Upx confers antiphage activity against ФV-1 infection but not against T6, Mu, P1, SP, T1, or vB phages. The authors should provide additional experimental evidence and analyses to clarify the mechanism underlying Upx-mediated phage resistance. Specifically, does ФV-1 possess a replication or transcription mode distinct from other phages that can be specifically targeted by the dual nucleases?\n2. The authors claim that both CTD and NTD exhibit sequence-independent 3’-5’ exonuclease activity based on electrophoresis gel analyses. However, further experiments are needed to validate this conclusion:\n(1) the degradation pattern of 5’-labeled ssDNA/ssRNA substrates suggests the presence of defined cleavage bands rather than a progressive degradation pattern typical of exonucleases.\n(2) the observed degradation products appear longer than single nucleotides. To clarify exonuclease activity, the authors should analyze cleavage products using both 5’- and 3’-labeled DNA/RNA substrates. If Upx functions as a true 3’-5’ exonuclease, only single-nucleotide products should be detected with 3’-labeled substrates. In Supplementary Figure 5d, the substrate appears resistant to cleavage by Upx and its domains, suggesting additional substrate requirements.\n(3) The authors should include DNA/RNA length markers in their gel analyses to accurately determine the length of cleavage products.\n(4) The authors should further investigate CTD/NTD cleavage using substrates of varying lengths and sequence compositions to define nuclease sequence specificity and cleavage-site preferences.\n(5) In Figure 4c, the labeled product should be \"ssRNA fragments\" rather than \"ssRNA fragment.\"\n3. In Supplementary Figure 2, class 1, 2, and 4 reconstructions contain additional densities adjacent to the NTD-MD-CTD region. The authors performed local refinement to obtain a high-resolution structure of Upx; however, these additional densities should not be disregarded. It is important to determine whether these densities correspond to Upx dimerization or oligomerization, co-purified substrates or potential interacting partners. A more detailed and careful structural analysis could provide valuable insights into Upx function.\n4. The most intriguing finding of this study is that Upx contains two distinct nuclease active sites—one constitutively active CTD and one autoinhibited NTD. However, key mechanistic questions remain unresolved. Is NTD activity essential for antiphage defense? A loss-of-function assay targeting NTD activity would help clarify its role. Second, the mechanism of NTD activation remains elusive. Further analysis is needed to elucidate how NTD is activated. While the authors tested several substrates in Supplementary Figure 5, the precise physiological substrate of Upx remains unknown. Given its substrate preference for ssRNA/ssDNA, one possibility is that Upx targets transcriptional R-loops, which contain both ssRNA and ssDNA.", "reviewer_2": "In their manuscript “Structural and Mechanistic Insights into the Dual-nuclease domain Defense 1 Protein as a Single-Molecule Anti-Phage System”, Zhou and colleagues report a cryo-EM structure of Upx. The structure shows that the protein has the previously identified PD-(D/E)XK domain at the C-terminus, a middle domain, and a PD-(D/E)XK like N-terminal domain, with a reduced number of beta sheets. The authors also report that the N-terminal domain has nuclease activity against single stranded DNA, and that the nuclease activity of the N- and C-terminal domains is controlled by the middle domain. The structures and their interpretation are satisfactory, but unfortunately, the structures provide little insight on how the anti-phage defense works, and how the specificity arises.\nCriticisms:\n• The authors report the experimental result that Upx28 provides defense against phage ФV-1 infection as a novel result. However, it was already known from prior work that the Upx28 defends against a relatively narrow spectrum of phages that includes ФV-1 (Gao et al., Science 2020).\n• The identification of the N-terminal domain as a nuclease domain lacks controls. Since the authors have identified the active site, they should make inactive mutants to prove that the reported nuclease activity is not due to a protein contaminant.\n• All evidence indicates that ФV-1 has a double stranded DNA genome. It therefore remains unclear what the ssDNA directed exonucleolytic activity of the N-terminal domain is directed against.\n• The transition from the nuclease-inactive conformation with middle domain mediated autoinhibition to an active conformation is presumably triggered by proteins of the phage (this may also explain the narrow spectrum of targeted phages. For the manuscript to have the impact required for Nature Comm., the authors would have to identify the trigger that converts the inactive to an active nuclease, and would have to explain how this change occurs.\nIn summary, the manuscript contains a cryo-EM structure and some biochemical data that could be a starting point for a more complete story on how the anti-phage protein Upx actually works, but at this point, the story is too incomplete to permit biological insight into how the anti-phage defense system actually works.", "reviewer_3": "In this manuscript, the authors provide a biochemical study of Upx, which they term DNDP. Using Cryo-EM and biochemical experiments, they define a second nuclease domain of Upx/DNDP, and provide preliminary evidence to suggest that the middle domain regulates the N-terminal and C-terminal nucleases. While exciting, the results feel incomplete. For Nature Communications, some additional data should be presented to validate the significance of the structural and biochemical work. The authors should not need to present a complete mechanism for Upx/DNDP for acceptance, but at current the manscript falls short of the standard of others published in this journal (a great example is https://www.nature.com/articles/s41467-025-58540-9 and https://www.nature.com/articles/s41467-025-57397-2 where structural, biochemical, and in vivo microbiological experiments were integrated to give a fuller picture of antiviral function). For this reason, I suggest the manuscript should be rejected or subject to major revisions to address several critical issues:\n1. The importance of biochemical results for phage defense is not examined. The authors identify several residues that are required for DNDP function and interactions between different domains which appear to regulate activity of different DNDP domains. None of these constructs are tested in the in vivo phage defense assay shown in Figure 1, which is critical to understanding whether different activities of DNDP are important for antiviral restriction. In particular, the importance of the NTD and interactions between MD and the NTD/CTD in defense against phage would be deeply interesting.\n2. The authors assert that DNDP is a nonspecific 3ʹ exonuclease that targets ssDNA and perhaps ssRNA. Their data reveal several potentially important aspects of the protein – a second nuclease domain (NTD), and a regulatory middle domain. In their different biochemical analyses, the enzyme appears to degrade substrates to a variety of different extents, and is most active at full-length. Given that DNDP seems to inhibit only very specific phages, the authors should more carefully examine its mechanism and the relative importance and role of different aspects of the structure they have discovered. Using DNA-seq or RNA-seq to examine activity of Upx/DNDP or its mutants in vivo would help address the importance of their biochemical results. Alternatively, using additional DNA and RNA substrates in vitro and quantitatively assessing those which are most preferred by the enzyme would help define the most important substrates. DNA or RNA degradation timecourses may also be helpful for confirming a nonspecific exonuclease activity.\n3. The importance/conservation of variant PD(D/E)XK nucleases like the NTD is touched on in the supplement. The authors should confirm they are descended from the PD(D/E)XK family using topology diagrams and/or structural trees (e.g. via foldseek). Further, they should consider showing that diverse homologs of these enzymes are functional by cloning/testing other potential phage defense systems containing these variant domains.\nMinor points:\n1. The authors state that the Upx gene was cloned from S. enterica NCTC6026. The original Upx system identified in Gao et al., 2020 was from E. coli NCTC8620. The authors should provide the exact Refseq accession or sequence of their cloned construct. It would appear that the gene cloned in this experiment matches at the protein level quite closely to the original Gao et al construct (judging by the sequence in the validation report). In the Gao et al., 2020 paper, Bacteriophage P1 was strongly restricted, but no P1 restriction is reported in this manuscript. It would be useful to clarify this point – is the construct the same?\n2. The authors state their construct is cloned into pBAD, and is thus presumably arabinose inducible. Was induction used in the experiments?\n3. Line 129-130: “These findings suggest the function of DNDP may be conserved across a large number of bacterial species.” How so? A single phage was restricted. Without more evidence (e.g. conservation of DNDP in different bacterial groups, etc), remove this statement.\n4. Fig 4B: A single band is evident after NTD cleavage. What is this product? Could this indicate function as an endonuclease under certain circumstances?\n5. Figure 3C: Error, “ssDNA fragment” should be ssRNA. Text states RNA oligo is 30 nt, but figure says 56 nt.\n6. Figure 3D: Legend states the gel is representing dsDNA cleavage experiments, but the data figure appears to concern metal dependence of the CTD. Please fix legend. Text references 3D to provide dsDNA cleavage data. Include data in figures.\n7. Figure 3D: Level of activity is low compared to data in 3B. Why?"}, "author_response": "Response to Comments\nReviewer #1 (Remarks to the Author):\nThe discovery and characterization of bacterial defense systems are under\nextensive investigation. In this study, Zhou, Wei, and colleagues\ncharacterized the Upx nuclease, revealing a unique dual-nuclease\narchitecture. As shown by biochemical and structural analysis, Upx contains\ntwo nuclease active sites: a classical C-terminal PD-(D/E)XK nuclease (CTD)\nand an atypical N-terminal PD-(D/E)XK nuclease (NTD). Functional analyses\nshow that the CTD can cleave both ssDNA and ssRNA, whereas the NTD is\nspecific for ssDNA. The authors further reported the cryo-EM structure of\napo Upx, providing structural insights into the active site features of\nboth NTD and CTD. Additionally, Upx harbors a middle domain (MD) that\ndifferentially regulates its nuclease activities—stimulating CTD while\ninhibiting NTD. While these findings are intriguing, additional experiments\nare required to fully elucidate the mechanism of Upx function.\nResponse: We thank the reviewer for the thoughtful summary of our work and\nfor recognizing the significance of uncovering the dual-nuclease\narchitecture of Upx. We fully agree that additional experiments are\nimportant to further elucidate the detailed mechanism of Upx function. In\nthe revised manuscript, we have substantially strengthened our study by:\n(i) Performing in vivo phage plaque assays with truncation and catalytic\nmutants, which demonstrate that both nuclease domains are indispensable for\nphage defense.\n(ii) Testing a broader panel of DNA and RNA substrates in vitro, along with\ntime-course assays and 3′-phosphorothioate protection experiments, to\nestablish substrate preferences and confirm the 3′-exonuclease activity.\n(iii) Conducting structural topology and phylogenetic analyses, along with\nfunctional validation of homologous SecC-containing nucleases, revealing\nthat these atypical PD-(D/E)XK enzymes are evolutionarily conserved and\nenzymatically active.\n(iv) Performing IP-mass spectrometry to identify interacting partners,\nincluding the phage DNA polymerase , which further supports the\nphysiological relevance of Upx’s substrates.\n(v) Discovering that gp16, a protein encoded by phage ΦV-1, stimulates the\nNTD-MD activity of Upx.\nThese new data establish a direct link between Upx’s biochemical\nactivities and phage defense, highlight the evolutionary conservation of\nthe dual-nuclease modules, and uncover a phage-encoded regulator of Upx\nactivity. Although we agree that the full mechanism of Upx is complex and\nmerits further investigation, we believe our current findings provide key\ninsights into the operation of this unique nuclease and lay a solid\nfoundation for future mechanistic studies.\n1. The authors demonstrate that Upx confers antiphage activity against ФV-\n1 infection but not against T6, Mu, P1, SP, T1, or vB phages. The authors\nshould provide additional experimental evidence and analyses to clarify the\nmechanism underlying Upx-mediated phage resistance. Specifically, does ФV-\n1 possess a replication or transcription mode distinct from other phages\nthat can be specifically targeted by the dual nucleases?\nResponse: We sincerely thank the reviewer for this insightful comment. To\ninvestigate the basis of Upx-mediated specificity toward phage ФV-1, we\nperformed immunoprecipitation followed by mass spectrometry (IP–MS) to\nidentify phage-derived interacting partners of Upx (Fig. R1a). Notably, we\ndiscovered that gp16, a protein encoded by phage ФV-1, strongly stimulates\nthe nuclease activity of the NTD–MD fragment of Upx (Fig. R1b, c). This\nfinding provides direct evidence that a phage-encoded factor can activate\nthe otherwise autoinhibited nuclease activity, thereby linking Upx activity\nto the infection process of ФV-1.\nTo evaluate whether gp16 is unique to ΦV-1, we conducted a BLAST search\nagainst the NCBI protein database and identified 941 homologous sequences.\nPhylogenetic analysis revealed that gp16 forms a distinct evolutionary\nbranch, with no close homologs detected in other phages (Fig. R1d).\nImportantly, none of these homologs were found in the six phages examined\nin our study that escape Upx restriction—including vB (Escherichia coli\nphage vB ECOM-KAW1E185), Mu, T6, P1, SP, and T1—and comparative genome\nanalyses confirmed the absence of gp16-like sequences in their genomes.\nThis absence of gp16 homologs in non-sensitive phages provides a plausible\nexplanation for their resistance to Upx activity.\nFurthermore, we systematically tested a wider panel of DNA and RNA\nsubstrates and found that Upx preferentially cleaves replication-associated\nDNA intermediates, such as forked DNA and D-loops, which are expected to\nform during active genome replication or transcription (Fig. R1e–l).\nConsistent with this, our IP–MS and pull-down assays revealed a direct\ninteraction between Upx and the phage DNA polymerase, supporting the notion\nthat D-loop–like replication intermediates represent the physiological\nsubstrates of Upx. These findings together suggest that Upx exerts its\nantiviral activity by targeting replication-associated DNA structures\nduring ΦV-1 infection.\nCollectively, our results support a model in which the ΦV-1–encoded\nprotein gp16 directly engages Upx and relieves its autoinhibition during\ninfection, thereby activating its nuclease activity toward replication- and\ntranscription-associated DNA intermediates. We have incorporated these new\ndata and mechanistic insights into the revised manuscript (Fig. R2), which\nsubstantially clarify the molecular basis of Upx-mediated phage\nspecificity.\nFig R1: The working model of DNDP in phage defense of ФV-1. (a) Schematic\nrepresentation of sample preparation for mass spectrometry analysis. (b)\nPulldown assay showing the interaction between DNDP and phage gp16. (c)\nActivation of NTD-MD-mediated cleavage of 5’-Cy3-labeled ssDNA by phage φ\nV-1 gp16. (d) Phylogenetic analysis of gp16 and its homologous proteins\nidentified in the NCBI database. gp16 is positioned on a distinct branch,\nindicating that no closely related homologs are present in other phages. (e-\ni) Cleavage activity of DNDP on different DNA substrates, including 5′\noverhang DNA, 3′ overhang DNA, partial duplex DNA, forked DNA. All cleavage\nproducts were analyzed by 13% denaturing urea–PAGE and visualized by\nfluorescence imaging. Each assay was performed in triplicate. (j) Cleavage\nassays were performed using the unpaired 3′ single-stranded regions within\nboth D-loop and R-loop structures. (k) Cleavage assays were performed using\nD-loop structures labeled with 5’ Cy3 at the terminus. The cleavage products\nwere analyzed by 6% native urea–PAGE and visualized by fluorescence imaging.\nEach assay was performed in triplicate.\nFig R2: Proposed working model of the DNDP-mediated antiphage defense system.\nIn the absence of phage, DNDP adopts an auto-inhibited conformation and\nexhibits only basal nuclease activity. Upon phage infection, the phage-\nencoded protein gp16 relieves MD-mediated inhibition, thereby enhancing NTD\nactivity to degrade phage-derived single-stranded DNA or RNA intermediates.\nThis degradation blocks phage genome replication and prevents phage\nproliferation.\n2. The authors claim that both CTD and NTD exhibit sequence-independent\n3’-5’ exonuclease activity based on electrophoresis gel analyses.\nHowever, further experiments are needed to validate this conclusion:\n(1) the degradation pattern of 5’-labeled ssDNA/ssRNA substrates suggests\nthe presence of defined cleavage bands rather than a progressive\ndegradation pattern typical of exonucleases.\nResponse: We sincerely appreciate the reviewer’s constructive suggestion.\nIn our initial experiments, the cleavage of 5′-labeled ssDNA and ssRNA\nsubstrates indeed appeared as discrete bands, which raised the possibility\nof defined endonucleolytic events. To clarify this, we carefully optimized\nboth reaction conditions and electrophoretic resolution. Specifically, assays\nwere performed with a protein-to-substrate molar ratio of 20:1 for 30 minutes\nat 37 ℃, and products were analyzed on a 13% urea–polyacrylamide gel,\ninitially run at 240 V for 5 minutes followed by 120 V for 1 hour. Under\nthese optimized conditions, Upx produced a continuous, progressive laddering\npattern for both ssDNA and ssRNA (Fig. R3a-d), which is characteristic of 3′\n–5′ exonuclease activity rather than discrete endonucleolytic cleavage. To\nfurther validate this conclusion, we employed 3’-phosphorothioate–modified\nsubstrates in which the last three nucleotides were protected. Under these\nconditions, cleavage was completely abolished, indicating that Upx strictly\nrequires a free 3’-hydroxyl group for catalysis (Fig. R3e). These data thus\nprovide stronger evidence supporting our conclusion that both the CTD and\nNTD of Upx function as bona fide exonuclease domains.\nFig. R3: ssDNA/ssRNA cleavage assays. (a) The 5’-Cy3-labeled 60 nt ssDNA was\nincubated with Upx, and cleavage assays were performed over a time course\nranging from 10 to 60 minutes. (b) The 3’-Cy3-labeled 60 nt ssDNA was\nincubated with Upx, and cleavage assays were performed over a time course\nranging from 10 to 60 minutes. (c) The 5’-Cy3-labeled 60 nt ssRNA was\nincubated with Upx, and cleavage assays were performed over a time course\nranging from 10 to 60 minutes. (d) The 3’-Cy3-labeled 60 nt ssRNA was\nincubated with Upx, and cleavage assays were performed over a time course\nranging from 10 to 60 minutes. (e) Cleavage of ssDNA substrates containing\nphosphorothioate modifications at the three terminal nucleotides of the 3’\nend by full-length Upx. All experiments were performed in triplicate on three\nindependent occasions.\n(2) the observed degradation products appear longer than single\nnucleotides. To clarify exonuclease activity, the authors should analyze\ncleavage products using both 5’- and 3’-labeled DNA/RNA substrates. If\nUpx functions as a true 3’-5’ exonuclease, only single-nucleotide\nproducts should be detected with 3’-labeled substrates. In Supplementary\nFigure 5d, the substrate appears resistant to cleavage by Upx and its domains,\nsuggesting additional substrate requirements.\nResponse: We sincerely thank the reviewer for this insightful comment. To\ndirectly address the concern, we examined the degradation products using both\n5’- and 3’-labeled ssDNA/ssRNA substrates (Fig. R4a-d). As expected for a\n3’–5’ exonuclease, no shorter fragments were detected from 3’-labeled\nsubstrates beyond the terminal nucleotide. The single nucleotide labeled with\nCy3 migrates in the opposite direction during electrophoresis due to the\npositive charge of the Cy3 dye. Consequently, the cleaved bands are observed\nat the upper region of the gel (Fig. R4a, b). It is possible that for the\nsame reason, the shorter cyanine-labeled oligonucleotides carry an anomalous\ncharge, leading to an upward shift of their bands during electrophoresis. To\nfurther validate this conclusion, we employed 3’-phosphorothioate–modified\nsubstrates in which the last three nucleotides were protected. Under these\nconditions, cleavage was completely abolished, indicating that Upx strictly\nrequires a free 3’-hydroxyl group for catalysis (Fig. R3e). These\ncomplementary experiments strongly support that Upx acts as a canonical 3’–\n5’ exonuclease rather than generating longer intermediate products through\nan alternative mechanism. We have revised Supplementary Figure 5 to clarify\nthis point and included the new supporting data.\nFig. R4: ssDNA/ssRNA cleavage assays. (a) The 5’-Cy3-labeled 60 nt ssDNA was\nincubated with Upx, and cleavage assays were performed over a time course\nranging from 10 to 60 minutes. The red arrow indicates the 5' Cy3-labeled\nsingle nucleotide. (b) The 3’-Cy3-labeled 60 nt ssDNA was incubated with\nUpx, and cleavage assays were performed over a time course ranging from 10\nto 60 minutes. The red arrow indicates the 5' Cy3-labeled single nucleotide.\n(3) The authors should include DNA/RNA length markers in their gel analyses\nto accurately determine the length of cleavage products.\nResponse: We thank the reviewer for this helpful suggestion. In the revised\nmanuscript, we have added appropriate single-stranded DNA markers to all\nrelevant denaturing gel figures (Fig. 3–5).\n(4) The authors should further investigate CTD/NTD cleavage using\nsubstrates of varying lengths and sequence compositions to define nuclease\nsequence specificity and cleavage-site preferences.\nResponse: We thank the reviewer for this valuable suggestion. We agree that\nassessing substrate preferences is important to better define Upx’s\nnuclease activity. To this end, we performed additional cleavage assays\nusing ssDNA substrates of varying lengths (60, 50, and 30 nt) (Fig. R5a,d).\nIn all cases, Upx consistently degraded the substrates without detectable\nsequence dependence. We further tested a series of truncated Upx variants\nagainst these ssDNA substrates and observed similar cleavage efficiencies,\nindicating that the nuclease activity is independent of both substrate\nlength and sequence composition (Fig. R5b–c). Consistent with our previous\ndata showing that Upx-mediated ssRNA cleavage also lacks sequence or length\ndependence (Fig. 3g and Fig. S4h, j, k), these results together demonstrate\nthat Upx functions as a sequence- and length-independent exonuclease.\nFig. R5: Cleavage assays of Upx using ssDNA substrates of varying lengths\nand sequences. (a) Cleavage assays of Upx using 5’ Cy3 labeled ssDNA\nsubstrates of varying lengths and sequences. (b-c) Cleavage assays were\nperformed with Upx and its truncated variants using two substrates: a 5' Cy3-\nlabeled 60-nt ssDNA (b) and a 5' Cy3-labeled 56-nt ssDNA (c). (d) The table\nof substrates sequences. All experiments were performed in triplicate on\nthree independent occasions.\n(5) In Figure 4c, the labeled product should be \"ssRNA fragments\" rather\nthan \"ssRNA fragment.\"\nResponse: We thank the reviewer for pointing out this detail. The figure has\nbeen updated with the corrected label, and all corresponding annotations have\nbeen revised to read “ssRNA fragments” accordingly.\n3. In Supplementary Figure 2, class 1, 2, and 4 reconstructions contain\nadditional densities adjacent to the NTD-MD-CTD region. The authors\nperformed local refinement to obtain a high-resolution structure of Upx;\nhowever, these additional densities should not be disregarded. It is\nimportant to determine whether these densities correspond to Upx\ndimerization or oligomerization, co-purified substrates or potential\ninteracting partners. A more detailed and careful structural analysis could\nprovide valuable insights into Upx function.\nResponse: We thank the reviewer for this valuable suggestion and fully\nagree that additional densities in cryo-EM reconstructions could provide\nimportant functional insights. To clarify their origin, we examined Upx\noligomeric status using both SEC and AUC, which consistently indicated that\nUpx exists as a monomer in solution (Fig. 1c and Fig. S1a). We also\nrepeated cryo-EM data collection at a lower sample concentration (1 mg/mL,\ncompared with 2.5 mg/mL previously). Under these conditions, the additional\ndensities were no longer observed (Fig. R6), suggesting that they most\nlikely arose from particle aggregation at high concentration rather than\nreflecting physiologically relevant dimerization or binding partners. We\nhave clarified this point in the revised manuscript.\nFig. R6: Supplementary Fig. 2: Cryo-EM reconstruction of DNDP protein. (a)\nA representative raw cryo-EM micrograph of the DNDP. (b) Representative 2D\nclass averages. (c) 3D classification. (d) Final local refinement without\nnoise. (e) Local resolution estimation of the reconstruction in (e). (f)\nFSC plots of the reconstruction in (e). (g) Angular distribution of the\nreconstruction in (e).\n4. The most intriguing finding of this study is that Upx contains two\ndistinct nuclease active sites—one constitutively active CTD and one\nautoinhibited NTD. However, key mechanistic questions remain unresolved. Is\nNTD activity essential for antiphage defense? A loss-of-function assay\ntargeting NTD activity would help clarify its role. Second, the mechanism\nof NTD activation remains elusive. Further analysis is needed to elucidate\nhow NTD is activated. While the authors tested several substrates in\nSupplementary Figure 5, the precise physiological substrate of Upx remains\nunknown. Given its substrate preference for ssRNA/ssDNA, one possibility is\nthat Upx targets transcriptional R-loops, which contain both ssRNA and\nssDNA.\nResponse: We thank the reviewer for these thoughtful and constructive comments.\nThe points raised—regarding the essential role of NTD activity in defense,\nthe mechanism of its activation, and the identification of physiological\nsubstrates—are central to understanding Upx function, and we have performed\nnew experiments to address each of them.\nTo directly test whether NTD activity is essential for antiphage defense, we\nperformed phage plaque assays using truncation and catalytic mutants. Our\nresults demonstrate that all three domains — NTD, MD, and CTD — are\nindispensable. Importantly, loss of NTD nuclease activity completely\nabrogates Upx's antiphage function (Fig. R7a, b).\nTo investigate the mechanism of NTD activation, we conducted co-\nimmunoprecipitation and pull-down assays coupled with mass spectrometry. This\nidentified several Upx-interacting proteins, including the phage-encoded gp16,\nwhich we found specifically activates the nuclease activity of the NTD–MD\nregion (Fig. R7c–e). Phylogenetic and comparative genomic analyses reveal\nthat gp16 forms a distinct evolutionary branch and is absent from the genomes\nof all Upx-insensitive phages tested. This narrow phylogenetic distribution\nprovides a mechanistic explanation for Upx's highly specific antiphage\nactivity, as its activation depends on a factor unique to a select group of\nphages including ΦV-1.\nFollowing the reviewer's suggestion, we also evaluated Upx activity on R-\nloops and D-loops. Upx cleaves both the unpaired 3′-ssDNA in D-loops and\nthe unpaired 3′-ssRNA in R-loops, with a preference for D-loop substrates\n(Fig. R7f). This indicates that Upx targets nucleic acid intermediates\ncontaining both ssDNA and ssRNA.\nCollectively, our new data establish that NTD activity is essential for\nantiphage defense, identify a phage protein that activates the\nautoinhibited NTD, and support a model in which Upx targets replication-\nand transcription-associated nucleic acid structures during phage\ninfection.\nFig R7: The NTD nuclease activity is essential for Upx defense and is\nactivated by the phage protein gp16. (a) Phage plaque assays of Upx truncation\nand point mutants against ФV-1 infection. Loss of the NTD or its catalytic\nresidues abolished defense. (b) Nuclease assays of NTD catalytic mutants on\n5′-Cy3–ssDNA. (c) Co-immunoprecipitation of DNDP with MBP-tagged gp16,\nshowing direct interaction between gp16 and Upx. (d) Pulldown assays of Upx\ntruncations with gp16, indicating that gp16 binds to the NTD–MD region. (e)\nNuclease assays showing that gp16 strongly stimulates the nuclease activity\nof NTD–MD. (f) Upx is capable of specifically and efficiently cleaving the\n3′-end unpaired ssDNA within D-loops, whereas its cleavage activity toward\nthe 3′-end unpaired ssRNA in R-loop is relatively weak. All experiments were\nperformed in triplicate on three independent occasions. All cleavage products\nwere analyzed by 13% denaturing urea–PAGE and visualized by fluorescence\nimaging. Each assay was performed in triplicate.\nReviewer #2 (Remarks to the Author):\nIn their manuscript “Structural and Mechanistic Insights into the Dual-\nnuclease domain Defense 1 Protein as a Single-Molecule Anti-Phage System”,\nZhou and colleagues report a cryo-EM structure of Upx. The structure shows\nthat the protein has the previously identified PD-(D/E)XK domain at the C-\nterminus, a middle domain, and a PD-(D/E)XK like N-terminal domain, with a\nreduced number of beta sheets. The authors also report that the N-terminal\ndomain has nuclease activity against single stranded DNA, and that the\nnuclease activity of the N- and C-terminal domains is controlled by the\nmiddle domain. The structures and their interpretation are satisfactory,\nbut unfortunately, the structures provide little insight on how the anti-\nphage defense works, and how the specificity arises.\nCriticisms:\n• The authors report the experimental result that Upx28 provides defense\nagainst phage ФV-1 infection as a novel result. However, it was already\nknown from prior work that the Upx28 defends against a relatively narrow\nspectrum of phages that includes ФV-1 (Gao et al., Science 2020).\nResponse: We thank the reviewer for raising this important point. We are\naware of the prior work by Gao et al. (Science 2020), which reported that\nUpx28 confers defense against a narrow spectrum of phages including ΦV-1.\nIn our manuscript, we did not intend to claim novelty in the phenotypic\nobservation of Upx-mediated Φ V-1 restriction. Instead, the central\ncontribution of our work lies in moving beyond the previously described\ndefense phenotype to elucidate the underlying molecular and structural\nmechanism. Through integrated structural, biochemical, and functional\nanalyses, we reveal that Upx possesses a unique dual-nuclease architecture,\nand we establish that both nuclease domains are indispensable for its\nantiphage activity.\nMoreover, our study now provides a mechanistic explanation for the narrow\nantiphage spectrum of Upx28. We identified that its activation depends on\nthe phage-encoded protein gp16, which is uniquely present in ΦV-1 and a very\nlimited set of related phages, but is absent in all non-sensitive phages\ntested. This finding directly links Upx28’s narrow defensive range to the\nphylogenetic distribution of its specific phage-encoded activator. Thus, our\nwork not only reveals the intrinsic dual-nuclease mechanism of Upx but also\nexplains the ecological and evolutionary basis for its phage specificity.\n• The identification of the N-terminal domain as a nuclease domain lacks\ncontrols. Since the authors have identified the active site, they should\nmake inactive mutants to prove that the reported nuclease activity is not\ndue to a protein contaminant.\nResponse: We thank the reviewer for this critical suggestion. To definitively\nconfirm that the nuclease activity originates from the Upx NTD rather than a\nco-purifying contaminant, we generated several catalytically inactive mutants\nby alanine substitution of key charged residues within the active site,\nincluding D127A/K129A, D112A, E147A, and D173A (Fig. R5). All of these mutants\nexhibited substantially reduced NTD nuclease activity, demonstrating that\nthese residues are essential for catalysis and confirming that the observed\nactivity is intrinsic to the NTD (Fig. R8).\nFig. R8: Mutations of key residues in the catalytic pocket of NTD inhibit\nssDNA cleavage. Assays were performed using 5′-Cy3–labeled ssDNA (60 nt)\nas substrate at a protein:substrate ratio of 20:1. All experiments were\nperformed in triplicate on three independent occasions.\n• All evidence indicates that ФV-1 has a double stranded DNA genome. It\ntherefore remains unclear what the ssDNA directed exonucleolytic activity\nof the N-terminal domain is directed against.\nResponse: We thank the reviewer for this insightful question, which addresses\na central aspect of Upx’s mechanism. We agree that the role of an ssDNA-\ndirected exonuclease during infection by a dsDNA phage requires clarification.\nOur data support a model in which Upx does not target the packaged dsDNA\ngenome, but rather degrades transient single-stranded nucleic acid\nintermediates generated during phage genome replication and transcription.\nThis model is strongly supported by our biochemical data. Cleavage assays\nusing structured nucleic acid substrates—including molecules with 3′ or 5′\noverhangs, forked DNA, D-loops, and R-loops—demonstrate that Upx as well as\nits NTD and CTD preferentially cleaves the 3′ ends of single-stranded regions\nwithin these architectures, which mimic intermediates formed during viral\nDNA replication and transcription (Fig. R9a-h). Specifically, Upx as well as\nits NTD and CTD efficiently cleaves both the ssDNA in D-loops\n(replication/recombination intermediates) and the ssRNA in R-loops\n(transcription intermediates), with a preference for D-loop substrates (Fig.\nR9g-h). This indicates that Upx can potentially target both replication and\ntranscription processes.\nFurthermore, co-immunoprecipitation followed by mass spectrometry and pull-\ndown assays revealed that Upx directly interacts with the phage DNA polymerase.\nThis interaction suggests the recruitment of Upx to active replication sites,\nreinforcing that replication intermediates are among its physiological\nsubstrates.\nCollectively, these findings support a mechanism in which Upx inhibits phage\npropagation by selectively degrading critical ssDNA/ssRNA intermediates\nessential for ΦV-1 genome replication and transcription, rather than the\nintact dsDNA genome itself.\nFig. R9: Cleavage assays using substrates with fluorescent labeled complex-\nstructured DNA. (a–e) Cleavage activity of DNDP on different DNA substrates,\nincluding 5′ overhang DNA, 3′ overhang DNA, partial duplex DNA, forked DNA.\n(f) Cleavage activity of DNDP on D-loop and R-loop structures. (g) Cleavage\nactivity of DNDP on different D-loop structures. The cleavage products were\nanalyzed by 6% native PAGE and visualized by fluorescence imaging. (h)\nPulldown assay showing direct interaction between DNDP and phage DNA\npolymerase (DNA-pol). All cleavage products except for native PAGE were\nresolved on 13% denaturing urea–PAGE and detected by fluorescence imaging.\nAll assays were conducted in triplicate.\n• The transition from the nuclease-inactive conformation with middle domain\nmediated autoinhibition to an active conformation is presumably triggered by\nproteins of the phage (this may also explain the narrow spectrum of targeted\nphages. For the manuscript to have the impact required for Nature Comm., the\nauthors would have to identify the trigger that converts the inactive to an\nactive nuclease, and would have to explain how this change occurs.\nResponse：We thank the reviewer for this insightful and constructive comment,\nwhich rightly points to the core mechanistic question of how Upx transitions\nfrom an autoinhibited to an active state. We fully agree that identifying\nthe physiological trigger of NTD activation is essential to understanding\nboth Upx’s mechanism and its narrow antiphage spectrum.\nIn direct response to this point, we have now identified the phage-encoded\nprotein gp16 as the key trigger that relieves the middle domain (MD)-mediated\nautoinhibition of the NTD. Through co-immunoprecipitation followed by mass\nspectrometry, and validated by pull-down assays, we found that gp16\nspecifically binds to Upx (Fig. R10b, c). Most importantly, subsequent\nnuclease assays demonstrated that gp16 strongly stimulates the ssDNA cleavage\nactivity of the NTD–MD fragment (Fig. R10d), providing direct functional\nevidence that gp16 acts as an activator that counteracts MD-mediated\ninhibition.\nThis finding not only identifies the specific phage factor responsible for\nactivating Upx but also provides a clear explanation for its narrow defense\nspectrum. As noted in our response to an earlier comment, phylogenetic\nanalysis confirms that gp16 is uniquely present in ΦV-1 and a small set of\nclosely related phages, and is absent in all Upx-insensitive phages tested.\nThus, Upx’s activity is contingent on the presence of this specific phage-\nencoded activator.\nTaken together, our results support a model in which Upx remains autoinhibited\nin the absence of infection. Upon ΦV-1 infection, the phage protein gp16\nbinds to the NTD–MD region, relieving autoinhibition and activating the NTD\nnuclease. This allows Upx to cleave replication- and transcription-associated\nnucleic acid intermediates, thereby executing antiphage defense. We are\nconfident that the identification of gp16 as the activation trigger\nsubstantially strengthens the mechanistic narrative of our study and meets\nthe level of insight expected for Nature Communications.\nFig. R10: Identification of phage φV-1 proteins interacting with DNDP. (a)\nPulldown assay showing direct interaction between DNDP and gp16. (b) gp16\nrelieves MD-mediated inhibition of NTD nuclease activity, assayed using 5’\nCy3-labeled ssDNA (60 nt). All cleavage products were analyzed by 13%\ndenaturing urea–PAGE and visualized by fluorescence imaging. Each assay was\nperformed in triplicate. (c) Phylogenetic analysis of gp16 and its homologous\nproteins identified in the NCBI database. gp16 is positioned on a distinct\nbranch, indicating that no closely related homologs are present in other\nphages. (d) Proposed working model of the DNDP-mediated antiphage defense\nsystem. In the absence of phage, DNDP adopts an auto-inhibited conformation\nand exhibits only basal nuclease activity. Upon phage infection, the phage-\nencoded protein gp16 relieves MD-mediated inhibition, thereby enhancing NTD\nactivity to degrade phage-derived single-stranded DNA or RNA intermediates.\nThis degradation blocks phage genome replication and prevents phage\nproliferation.\nIn summary, the manuscript contains a cryo-EM structure and some biochemical\ndata that could be a starting point for a more complete story on how the\nanti-phage protein Upx actually works, but at this point, the story is too\nincomplete to permit biological insight into how the anti-phage defense system\nactually works.\nResponse: We thank the reviewer for this overall assessment of our initial\nsubmission. We agree that the original manuscript, while presenting a novel\nstructure and basic biochemical characterization, left important mechanistic\nquestions unanswered. However, in response to the reviewer's specific\ncritiques throughout this review process, we have conducted substantial new\nexperimentation that now provides a much more complete and biologically\ninsightful story.\nThe revised manuscript moves decisively beyond the starting point\nacknowledged by the reviewer by delivering three key mechanistic advances:\n1) Identification of the activation trigger: We have discovered that the\nphage-encoded protein gp16 is the specific trigger that relieves the\nmiddle domain-mediated autoinhibition of the NTD. This is not merely an\ninteraction; functional nuclease assays demonstrate that gp16 directly\nand strongly stimulates the activity of the autoinhibited NTD–MD fragment,\nanswering the fundamental question of how Upx is activated during phage\ninfection.\n2) Elucidation of the specificity mechanism: We provide a clear explanation\nfor Upx's narrow antiphage spectrum by showing that the activator gp16 is\nphylogenetically restricted. It is uniquely found in ΦV-1 and a narrow\nset of related phages and is absent from the genomes of all non-sensitive\nphages tested. This directly links Upx's activation and defensive\ncapability to the presence of a specific, phage-encoded co-factor.\n3) Definition of physiological function and substrates: We have significantly\nstrengthened the model of what Upx targets. Through comprehensive cleavage\nassays, we show that Upx preferentially degrades structured nucleic acid\nintermediates—most notably D-loops and replication forks—that arise\nduring phage genome replication or recombination. Furthermore, its direct\ninteraction with the phage DNA polymerase, revealed by IP-MS, supports\nits recruitment to replication sites in vivo. These data strongly argue\nthat Upx halts phage propagation by degrading essential ssDNA\nintermediates of replication, not the packaged dsDNA genome.\nCollectively, these new findings allow us to propose a coherent and well-\nsupported model: Upx remains autoinhibited until the infecting phage ΦV-1\nproduces gp16, which binds to and activates the NTD, enabling the degradation\nof phage replication intermediates and aborting the infection.\nWe are confident that these extensive additions, undertaken directly in\nresponse to the reviewer's insightful comments, have transformed the\nmanuscript from a preliminary characterization into a mechanistically\ngrounded study that offers substantial biological insight into the operation\nand specificity of a unique dual-nuclease defense system. We believe it now\nmeets the standard of mechanistic depth expected for Nature Communications.\nReviewer #3 (Remarks to the Author):\nIn this manuscript, the authors provide a biochemical study of Upx, which\nthey term DNDP. Using Cryo-EM and biochemical experiments, they define a\nsecond nuclease domain of Upx/DNDP, and provide preliminary evidence to\nsuggest that the middle domain regulates the N-terminal and C-terminal\nnucleases. While exciting, the results feel incomplete. For Nature\nCommunications, some additional data should be presented to validate the\nsignificance of the structural and biochemical work. The authors should not\nneed to present a complete mechanism for Upx/DNDP for acceptance, but at\ncurrent the manscript falls short of the standard of others published in this\njournal (a great example is https://www.nature.com/articles/s41467-025-\n58540-9 and https://www.nature.com/articles/s41467-025-57397-2 where\nstructural, biochemical, and in vivo microbiological experiments were\nintegrated to give a fuller picture of antiviral function). For this reason,\nI suggest the manuscript should be rejected or subject to major revisions to\naddress several critical issues:\nResponse: We thank the reviewer for their thoughtful assessment and for\nhighlighting the importance of integrating structural, biochemical, and\nmicrobiological data to build a compelling narrative. We agree that the\ninitial version of the manuscript, while establishing the unique dual-\nnuclease architecture of Upx, did not fully bridge the gap to its biological\nfunction in antiphage defense.\nIn direct response to this critique and the reviewer's valuable suggestions,\nwe have undertaken a substantial and comprehensive set of new experiments.\nThese additions have fundamentally transformed the manuscript by providing\nthe critical mechanistic and functional insights that were previously missing.\nOur revised study now delivers:\n1) Definitive functional validation: We performed essential in vivo phage\nplaque assays using a series of truncation and catalytic point mutants.\nThese experiments conclusively demonstrate that both the N-terminal and\nC-terminal nuclease domains are indispensable for Upx's antiphage activity,\nfirmly establishing the biological relevance of the dual-nuclease\narchitecture.\n2) Discovery of the activation trigger and specificity determinant: Through\nco-immunoprecipitation coupled with mass spectrometry and subsequent\nvalidation by pull-down and nuclease assays, we identified the phage-\nencoded protein gp16 as the key activator that relieves the middle domain-\nmediated autoinhibition of the NTD. Furthermore, phylogenetic analysis\nreveals that gp16 is uniquely present in ΦV-1 and absent in resistant\nphages, providing a clear evolutionary and mechanistic explanation for\nUpx's narrow defense spectrum.\n3) Identification of physiological context and substrates: We significantly\nadvanced the model of what Upx targets in vivo. By testing a panel of\nstructured nucleic acid substrates, we show that Upx preferentially\ncleaves replication and recombination intermediates, such as D-loops and\nforked DNA. Crucially, our discovery of a direct interaction between Upx\nand the phage DNA polymerase strongly supports its recruitment to active\nreplication sites, painting a clear picture of its physiological function\nin disrupting phage genome replication.\nCollectively, these new data allow us to propose a coherent and well-supported\nmechanistic model: Upx is maintained in an autoinhibited state until phage\ninfection; the production of the specific phage protein gp16 then activates\nUpx by binding to the NTD-MD region; the activated nuclease is recruited to\nreplication forks where it degrades essential single-stranded DNA\nintermediates, thereby aborting the phage life cycle.\nWe are confident that these extensive revisions, which seamlessly integrate\ncryo-EM structure, rigorous biochemistry, and decisive microbiological assays,\nhave elevated the manuscript to meet the standard of mechanistic insight and\nbiological significance expected by Nature Communications. We believe the\nwork now provides a complete and compelling story on the function of this\nunique dual-nuclease defense system.\n1. The importance of biochemical results for phage defense is not examined.\nThe authors identify several residues that are required for DNDP function\nand interactions between different domains which appear to regulate activity\nof different DNDP domains. None of these constructs are tested in the in vivo\nphage defense assay shown in Figure 1, which is critical to understanding\nwhether different activities of DNDP are important for antiviral restriction.\nIn particular, the importance of the NTD and interactions between MD and the\nNTD/CTD in defense against phage would be deeply interesting.\nResponse: We thank the reviewer for raising this important point. We fully\nagree that it is critical to connect the biochemical activities of DNDP with\ntheir relevance in phage defense. To directly address this, we performed in\nvivo phage plaque assays using both truncation constructs and catalytic point\nmutants (Fig. R11a). The results unequivocally demonstrate that all three\ndomains are indispensable for defense: deletion of either the NTD or CTD, or\nloss of catalytic activity in either nuclease domain, completely abolished\nantiphage activity. This confirms that both nuclease domains are functionally\nessential in the physiological context. Furthermore, we systematically\nevaluated a set of amino acid substitutions at key residues involved in NTD–\nMD and MD–CTD interactions. While these mutations perturbed inter-domain\nregulation in vitro, they did not compromise antiphage defense in vivo (Fig.\nR11b). This suggests that the specific inter-domain contacts we identified\nare not strictly required for restriction under these conditions, and that\nDNDP may employ additional or redundant interaction mechanisms in cells to\nensure functional robustness. Taken together, these findings directly link\nthe biochemical and biological functions of DNDP, demonstrating that while\ncatalytic residues are indispensable for defense, inter-domain regulatory\ninteractions may play a modulatory rather than essential role in vivo.\nFig. R11: All domains of DNDP are essential for antiphage defense. (a–b)\nPlaque assays showing the antiphage activity of wild-type DNDP and its\ntruncation ( Δ CTD, Δ NTD) or point-mutant variants (D127A/R129A,\nE1176A/K1178A, R530A/E562A/R571A, R707A/R595A/R541A/N705A) against phage Φ\nV-1 infection. Serial dilutions of phage lysates (10⁻¹ to 10⁻⁸) were plated\non E. coli MG1655 expressing the indicated constructs.\n2. The authors assert that DNDP is a nonspecific 3ʹ exonuclease that targets\nssDNA and perhaps ssRNA. Their data reveal several potentially important\naspects of the protein – a second nuclease domain (NTD), and a regulatory\nmiddle domain. In their different biochemical analyses, the enzyme appears\nto degrade substrates to a variety of different extents, and is most active\nat full-length. Given that DNDP seems to inhibit only very specific phages,\nthe authors should more carefully examine its mechanism and the relative\nimportance and role of different aspects of the structure they have discovered.\nUsing DNA-seq or RNA-seq to examine activity of Upx/DNDP or its mutants in\nvivo would help address the importance of their biochemical results.\nAlternatively, using additional DNA and RNA substrates in vitro and\nquantitatively assessing those which are most preferred by the enzyme would\nhelp define the most important substrates. DNA or RNA degradation timecourses\nmay also be helpful for confirming a nonspecific exonuclease activity.\nResponse: We thank the reviewer for these constructive suggestions regarding\nthe need to more precisely define DNDP's nuclease mechanism and substrate\npreferences. In response, we have conducted a comprehensive series of new\nexperiments that substantially strengthen our mechanistic conclusions.\nTo firmly establish the biological relevance of our biochemical findings, we\nfirst performed in vivo phage plaque assays using truncation and catalytic\nmutants. These experiments unequivocally demonstrated that both nuclease\ndomains are indispensable for antiphage defense (Fig. R12a).\nWe next systematically characterized DNDP's enzymatic properties through an\nexpanded panel of biochemical assays. Time-course experiments confirmed a\nprogressive 3′→5′ degradation pattern for both ssDNA and ssRNA, with a\nclear preference for ssDNA substrates (Fig. R12b, c). The absolute requirement\nfor a free 3′-hydroxyl group was established using 3′-phosphorothioate-\nblocked substrates, which completely abolished cleavage activity (Fig. R12d).\nFurther analysis with substrates of varying lengths and sequences reinforced\nthat DNDP functions as a sequence-independent exonuclease (Fig. R12e).\nMost critically, we quantitatively evaluated DNDP's activity against a broad\nspectrum of structured nucleic acid substrates—including various overhang\nconfigurations, forked DNA, D-loops, and R-loops (Fig. R12f–l). These assays\nrevealed robust cleavage of replication-associated intermediates such as D-\nloops and forked DNA, while activity toward R-loop substrates was\ncomparatively weaker. This distinct substrate preference profile strongly\nsupports our model that DNDP primarily targets replication or recombination\nintermediates during phage infection.\nCollectively, these comprehensive analyses provide compelling evidence that\nDNDP functions as a non-sequence-specific 3′→5′ exonuclease with a defined\npreference for replication-associated DNA intermediates, thereby elucidating\nthe molecular basis of its antiphase activity against ΦV-1.\nFig. R12: Cleavage assays using ssDNA and ssRNA substrates with both 5’- and\n3’-labeled DNA/RNA substrates. (a) Plaque assays showing the antiphage\nactivity of wild-type DNDP and its truncation (ΔCTD, ΔNTD) or point-mutant\nvariants (D127A/R129A, E1176A/K1178A) against phage ΦV-1 infection. Serial\ndilutions of phage lysates (10⁻¹ to 10⁻⁸) were plated on E. coli MG1655\nexpressing the indicated constructs. (b) 5’ Cy3-ssDNA (60 nt) time course\ncleavage assays with Upx. (c) 5’ Cy3-ssRNA (45 nt) time course cleavage\nassays with Upx. (d) Cleavage assays using 5’ Cy3-ssDNA substrates with\nphosphorothioate modifications at the three 5’-terminal nucleotides, tested\nwith full-length Upx, its NTD and CTD. (e) Biochemical cleavage assays using\nssDNA substrates of varying lengths and sequences. (f–j) Cleavage activity\nof DNDP on different DNA substrates, including 5′ overhang DNA, 3′ overhang\nDNA, partial duplex DNA, forked DNA. All cleavage products were analyzed by\n13% denaturing urea–PAGE and visualized by fluorescence imaging. Each assay\nwas performed in triplicate. (k) Cleavage assays were performed using D-loop\nstructures labeled with 5’ Cy3 at the terminus. The cleavage products were\nanalyzed by 6% native urea–PAGE and visualized by fluorescence imaging. Each\nassay was performed in triplicate. (l) Upx is capable of specifically and\nefficiently cleaving the 3′-end unpaired ssDNA within D-loop, whereas its\ncleavage activity toward the 3′-end unpaired ssRNA in R-loop is relatively\nweak. All experiments were performed in triplicate on three independent\noccasions. All cleavage products were analyzed by 13% denaturing urea–PAGE\nand visualized by fluorescence imaging. Each assay was performed in triplicate.\n3. The importance/conservation of variant PD(D/E)XK nucleases like the NTD\nis touched on in the supplement. The authors should confirm they are descended\nfrom the PD(D/E)XK family using topology diagrams and/or structural trees\n(e.g. via foldseek). Further, they should consider showing that diverse\nhomologs of these enzymes are functional by cloning/testing other potential\nphage defense systems containing these variant domains.\nResponse: We thank the reviewer for this constructive suggestion regarding\nthe evolutionary and functional analysis of the variant PD(D/E)XK nucleases.\nIn response, we have performed comprehensive structural and phylogenetic\nanalyses to rigorously establish the evolutionary origin of the DNDP NTD.\nFirst, detailed structural topology diagrams confirm that the NTD adopts a\ncanonical PD(D/E)XK fold, despite its reduced β-sheet content and sequence\ndivergence (Fig. R13a). To further trace its evolutionary lineage, we\nconducted structural similarity-based phylogenetic analysis using Foldseek.\nThis analysis robustly places the NTD within the broader PD(D/E)XK nuclease\nsuperfamily, revealing that it forms a distinct, divergent clade that branched\noff from classical PD(D/E)XK nucleases (Fig. R13b). To directly test whether\nthese evolutionarily divergent domains retain nuclease function, we cloned,\nexpressed, and purified homologous proteins from several distinct bacterial\nspecies that contain SecC-associated variant PD(D/E)XK domains. Biochemical\nassays demonstrated that these homologs exhibit robust 3′→5′ exonuclease\nactivity against ssDNA substrates (Fig. R13c, d), confirming that the\nenzymatic function is conserved across this variant family. Together, these\nintegrated structural, phylogenetic, and functional analyses firmly establish\nthat the NTD and its homologs represent a functional, evolutionarily conserved\nlineage derived from the PD(D/E)XK nuclease superfamily.\nFig. R13: (a) Topological comparison of the classical PD-(D/E)XK fold with\nthe catalytic cores of Upx-CTD, Upx-NTD, and SecC. (b) Phylogenetic analysis\nof 152 protein sequences reveals a SecC-containing clade. (c) Effects of\nmutating key catalytic residues (D122A/R129A, D127A, E147A, D173A,\nK111A/K134A, D135A/N138A) on nuclease activity, assayed using 5’ Cy3-labeled\nssDNA (60 nt) at a protein-to-substrate ratio of 20:1. (d) Time-course\nanalysis of SecC nuclease activity on 5’ Cy3-labeled ssDNA (60 nt).\nMinor points:\n1. The authors state that the Upx gene was cloned from S. enterica NCTC6026.\nThe original Upx system identified in Gao et al., 2020 was from E. coli\nNCTC8620. The authors should provide the exact Refseq accession or sequence\nof their cloned construct. It would appear that the gene cloned in this\nexperiment matches at the protein level quite closely to the original Gao et\nal construct (judging by the sequence in the validation report). In the Gao\net al., 2020 paper, Bacteriophage P1 was strongly restricted, but no P1\nrestriction is reported in this manuscript. It would be useful to clarify\nthis point – is the construct the same?\nResponse: We thank the reviewer for noting the origin and specificity of our\nUpx construct and the discrepancy with Gao et al., 2020. Our Upx gene was\ncloned from Salmonella enterica NCTC6026 (NCTC6026_01102, protein\nSQI81149.1), which shares 78.5% protein identity with the E. coli ortholog\n(WP_218067149.1) (Fig. R14). The observed difference in P1 restriction is\nlikely due to sequence differences. We have clarified this point in the\nrevised discussion.\nFig. R14: Multiple-sequence alignment of SeUpx and EcUpx generated with T-\nCoffee.\n2. The authors state their construct is cloned into pBAD, and is thus\npresumably arabinose inducible. Was induction used in the experiments?\nResponse: We thank the reviewer for highlighting the importance of induction.\nGene expression from the pBAD vector was consistently induced with 0.2% (w/v)\nL-arabinose in all experiments, including liquid cultures, LB agar plates,\nand soft agar overlays, ensuring high-level expression during phage challenge.\nWe have revised the Materials and Methods section to clearly state this for\nreproducibility.\n3. Line 129-130: “These findings suggest the function of DNDP may be\nconserved across a large number of bacterial species.” How so? A single\nphage was restricted. Without more evidence (e.g. conservation of DNDP in\ndifferent bacterial groups, etc), remove this statement.\nResponse: We appreciate the reviewer’ s suggestion. The sentence regarding\nbroad conservation of DNDP function has been removed. The paragraph now\nconcludes: “These results demonstrate that the DNDP protein confers defense\nagainst phage ΦV-1 infection in E. coli.” This revision ensures that our\nclaims remain strictly evidence-based.\n4. Fig 4B: A single band is evident after NTD cleavage. What is this product?\nCould this indicate function as an endonuclease under certain circumstances?\nResponse: We thank the reviewer for noting the single band in Figure 4B and\nsuggesting possible endonucleolytic activity. We repeated the experiment\nunder optimized conditions, and the revised figure now shows a ladder-like\npattern of degradation products, consistent with exonuclease activity. This\nconfirms that the enzyme functions as an exonuclease, and the text and legend\nhave been updated accordingly. We appreciate the reviewer’s suggestion, which\nhas strengthened our mechanistic conclusion.\n5. Figure 3C: Error, “ssDNA fragment” should be ssRNA. Text states RNA\noligo is 30 nt, but figure says 56 nt.\nResponse: We thank the reviewer for catching this error. The figure has been\nreplaced with the corrected version, and all labels and annotations have been\nfixed accordingly.\n6. Figure 3D: Legend states the gel is representing dsDNA cleavage experiments,\nbut the data figure appears to concern metal dependence of the CTD. Please\nfix legend. Text references 3D to provide dsDNA cleavage data. Include data\nin figures.\nResponse: We apologize for this error. The figure legend has been corrected,\nand the missing dsDNA cleavage data are now included in the Supplementary\nInformation. Corresponding legends and text have also been revised for\nconsistency.\n7. Figure 3D: Level of activity is low compared to data in 3B. Why?\nResponse: We thank the reviewer for highlighting this discrepancy. Upon re-\nexamining our data, we found that the experiments in Figures 3B and 3D were\nperformed with different protein preparations and substrate batches, which\nled to the difference in absolute activity levels. To address this\nconclusively, we have repeated all experiments for Figure 3B and 3D side-by-\nside in a single, unified experiment using the same master mix of protein\nand substrate. The new results, now presented in the revised figures, show\nconsistent activity levels across all panels.\nResponse to Comments\nReviewer#1(Remarks to theAuthor):\nTheauthorsaddressed allmycomments.\nResponse:Wesincerelythankyouforyour positiveevaluationofourrevised manuscript\nandforconfirmingthatallofyourpreviouscomments havebeensatisfactorily\naddressed.Wearegratefulforyourthoughtfulfeedbackthroughoutthereviewprocess,\nwhichhassubstantiallyenhanced theclarityand overallqualityofourwork.\nReviewer#2(Remarks to theAuthor):\nIwasverycriticalofthismanuscriptearlier,becauseIfeltthatthestructuralworkdidnot\nprovidesufficientinsightinto themechanismofaction oftheDual-nucleasedomain\nDefenseProtein(DNDP).Theauthors havenowpreparedavastlyimprovedversionof\nthemanuscriptwith keynewinsights.\n-withthephageencoded gp16, theauthorshaveidentifiedthetriggerforDNDP\nnucleaseactivation.Althoughtheactivationeffectisnotsuper-strong,theexplanationis\nlikelycorrect,becausethenarrowphylogenetic distributionofgp16 nicelyexplainsthe\nnarrowphagespecificityofthenewdefensesystem.\n-withtheirdemonstrationthattheDNDPisrecruited to thephagepolymerase, the\nauthorsalsoprovideaconvincing rationaleforhow DNDPactivityagainstsingle\nstrandednucleicacid contributestotheanti-phagedefense.\nThenewinsightsmakethemanuscriptNatureComm.material inmyeyes.\nReponse:Wesincerelyappreciateyourthoughtfulreassessmentofourrevised\nmanuscriptandaregratefulforyourrecognition ofthekeynewmechanisticinsights we\nhaveadded.Yourearliercriticalfeedbackplayed acrucial roleinguidingthese\nimprovements,particularlyinrefiningourstructuralandmechanistic analyses ofUpx\nfunction.Wearedelighted thattheaddedevidence hasaddressed yourconcernsand\nclarified themolecularbasisofUpx-mediated defense.Thankyouagainforyour\nconstructivecritiqueandforacknowledgingthestrengthened qualityofourwork.\nReviewer#3(Remarks to theAuthor):\nReviewofZhou etal.,forNatureCommunications:\nThismanuscript byZhou,Liu,Zhang,andYinetal.,presentsstructuralandfunctional\ndataandseeks todeterminethemechanismofUpx immunityinbacteria.Thisrevised\nmanuscriptaddressesseveralofthemajorissuesthatwereraisedafterinitialsubmission.\nNamely,theauthorshavenowmadestructure-guided mutationsandassessed their\nbiochemicalconsequencesas wellastheimpactofthese mutationson phagedefense.\nImportantly,theyshowthatallofthedomainsandactivesiteresidues identifiedare\nrequiredfordefense.BiochemicalexperimentsshowthatUpx/DNDPhasapreference\nforcleavageatthe3’ endofDNA,hintingathowUpx mightdefendagainstphagein\nvivo.Finally,theauthorsused IP-MSinanattempt to identifyphagefactorsthat may\nactivateorinfluence Upxfunctioninvivo.Overall,themanuscriptissignificantly\nimproved,buttheinclusion thesenewobservations nowraisessomeadditional\nquestionswhichshouldbeaddressed priortopublication.\nReponse：Wesincerelythankyouforyourthoroughevaluation ofourrevised\nmanuscriptandforrecognizingthesubstantialimprovementsmadeinresponseto the\ninitialreview.Wegreatlyappreciateyouracknowledgementthatthestructuralanalysis,\nstructure-guidedmutagenesis,biochemicalcharacterization,andphage-defense assays\ncollectivelystrengthenthemechanistic understandingofUpx.Weare gratefulforyour\nconstructiveandinsightfulfeedback,andweaddresseachofyourpointsindetailbelow.\nMajorpoints:\n-TheauthorsperformedIP-MSandidentifiedgp16and thephageDNApolymeraseas\naninteraction partnersofUpx/DNDP,andin themethods theystate thatlabel-free\nquantificationwasused.Thesedatashould beprovidedinatableorasamain-text\nfigureinthepaper.Second,theyshowthatgp16seems to potentiatetheactivityofan\nNTD-MDfusion,buttheydonotshowdataforfull-length Upx/DNDP.Doesitalso\nactivatethefull-length,WT protein?IsUpx actinglikean abortive-infectionsystem?\nDoesexpressionofgp16in theabsenceofphageinfectionresultintoxicity?This\nexperimentwouldseemcrucialto helpdelineatethebestmodelforthedata,positiveor\nnegativein result.Further,theauthorsdo notdiscusswhatgp16is,andwhatitsroleis\nduringphageinfection,andthereisnodiscussionofwhyitmightactivateUpx.Gp16isa\nlarge,multimericstructuralcomponentofthephagevirion. Thiswould seempertinent\nto discuss.\nResponse:Wethankthereviewerforthese insightfulcomments.Inresponse, wehave\nperformedadditionalexperimentsandsubstantiallyexpanded boththedata\npresentationandmechanistic discussionto directlyaddressallaspectsofthisimportant\npoint.\n(i)IP-MSlabel-freequantificationdata\nAssuggested,wenowprovidethecompletelist ofphageproteinsidentifiedby\nlabel-freemassspectrometryinSupplementaryTable3.Thistableincludesquantitative\nenrichmentvaluesforgp16, thephageDNApolymerase, and allotherphageproteins\ndetectedinourIP-MSexperiments.\n(ii) Activationoffull-length Upx/DNDPbygp16\nInadditionto theNTD–MDfusionassayshownpreviously,wehavenowdirectlytested\nwhethergp16canstimulatethenuclease activityofthefull-length wild-typeUpx\nprotein.Thenewbiochemicaldatademonstrate thatgp16indeed enhancesthe\nnucleaseactivityoffull-length Upx,consistentwithitseffectontheNTD–MDfragment.\nThesenewresultshavebeenincorporatedintotherevisedmanuscriptasSupplementary\nFigure6f (Fig.R1a).\n(iii)Evidencesupportingabortive-infection–likebehavior\nToclarifywhetherUpxfunctionsinamanneranalogoustoan abortive-infectionsystem,\nweperformed phage-infectionassays acrossarangeofMOIs.Weobservedthat\nincreasingphagedoseprogressivelysuppressed bacterial growth.However,even at\nhighMOIs,thecultureseventuallyrecovered,indicatingthatUpxeffectivelyprevents\nphageamplificationthroughanabortive-infection–likemechanism.In thissystem,\ninfectedcellslikelyundergogrowtharrestordeathtoblockphagepropagation,thereby\nprotectingtheremainingbacterialpopulation.TheseresultsarenowpresentedasFigure\n7c (FigR1b) anddiscussedintherevisedmanuscript.Thecorresponding textnowreads:\n“Toelucidatethesystem-leveldefensestrategyofUpx,weinvestigatedwhetherit\nfunctionsviaanabortive-infectionmechanism.Phage-infectionassaysacrossarangeof\nmultiplicitiesofinfection(MOIs)revealedthatwhileincreasingphagedosesinitially\nsuppressedbacterialgrowth,theculturesconsistentlyrecoveredovertime(Fig.7c).This\nrecoverypattern,wherethebacterialpopulationsurvivesdespiteinitialinfection,\nindicatesthatUpxpreventssuccessfulphageamplification.Thisisconsistentwithan\nabortive-infection–likemechanism,whereininfectedcellslikelyundergogrowtharrest\nordeathtoblockphagepropagation,therebyprotectingtheoverallbacterial\npopulation.“\n(iv) gp16 expressiondoesnotcausetoxicityintheabsenceofphage\nTodeterminewhethergp16 aloneistoxicto cells,weconductedcolony-formation\nassayswith ectopicgp16expression.Asshown inrevisedSupplementaryFigure6g (Fig\nR1c),nosignificantcytotoxicitywas observed forgp16compared to emptyvector\ncontrols.\n(v)Discussionofthebiologicalroleofgp16\nWehaveexpandedthediscussionofgp16'sbiologicalcontextatits firstintroductionin\nthetext(page7-8), whichnowreads:"}, {"round": 2, "reviewer_comments": {"reviewer_3": "I appreciate how the authors have addressed my comments, and I feel the manuscript is much improved as a result. I thank them very much for their care, and look forward to seeing the final published result.\nI have two final minor comments:\n- The authors should clarify how their DNA-seq data were collected and analyzed– the DNA extraction and bioinformatic analysis for these data are absent from the methods. In particular, how are these data being normalized? The legend states they are normalized to a control, but the control normalized read depth is not 1, so I don’t understand.\n- The authors now present a model wherein Upx functions as an abortive-infection system. This would imply that gp16 triggers Upx-mediated toxicity in the absence of phage infection. They have included data showing that gp16 itself is not toxic, but these data do not address the question of whether gp16 is an in vivo trigger of Upx-mediated toxicity (e.g. abortive infection). My previous review was poorly worded, and I apologize for this, but the question at hand is whether gp16 activates Upx and the new data do not address this. However, the new data included in Figure 7C showing a growth curve of cells expressing Upx infected at different MOIs shows a phenotype similar to those for direct defense systems in the literature, not an abortive system in my view. I suggest the authors review Vasallo et al., 2022 Figure 3B and Extended Data Figure 3 and other manuscripts on direct defense systems. If the authors agree that Upx is performing like a direct system, I would suggest revising the text. The gp16 toxicity data by themselves are unfortunately not that meaningful. They could either be removed, or replaced with an experiment testing whether gp16 triggers Upx-mediated toxicity in a two-plasmid system. If it does not, this would further agree with the concept that Upx is a direct system wherein gp16 may play a role in facilitating activity in vivo. However, I don’t think this toxicity experiment is essential in light of the MOI curve in 7C."}, "author_response": "Response to Comments\nReviewer #3 (Remarks to the Author):\nI appreciate how the authors have addressed my comments, and I feel the manuscript is\nmuch improved as a result. I thank them very much for their care, and look forward to seeing\nthe final published result.\nResponse: We sincerely thank you for your positive evaluation of our revised manuscript and\nfor acknowledging the improvements made in response to your previous comments. We are\ngrateful for your thoughtful feedback throughout the review process, which has substantially\nenhanced the quality of our work.\nI have two final minor comments:\n- The authors should clarify how their DNA-seq data were collected and analyzed– the DNA\nextraction and bioinformatic analysis for these data are absent from the methods. In\nparticular, how are these data being normalized? The legend states they are normalized to a\ncontrol, but the control normalized read depth is not 1, so I don’t understand.\nResponse: We thank the reviewer for raising these important points and sincerely apologize\nfor the initial omission of the DNA-seq methodology. As requested, we have now added a\ndetailed section to the Methods.\nIn brief, genomic DNA was extracted from infected E. coli cultures (5/20 min post-infection,\nMOI=3) using a commercial kit. Libraries were constructed and sequenced on Illumina\nplatforms. Bioinformatic processing involved quality control (fastp), alignment to a combined\nE. coli–ФV-1 reference genome (Sentieon pipeline), and read-depth (RD) analysis. To\nquantify phage DNA abundance, our RD-based approach follows established principles for\ncopy-number analysis, as implemented in tools such as CNVnator (Abyzov et al., Genome\nRes. 2011), which normalizes read counts against a sample-internal genomic baseline.\nRegarding the specific question about normalization, we have revised the text and figure\nlegend to clarify. For each sample, the RD was normalized to the genomic average RD across\nthe entire combined reference genome, establishing a sample-specific baseline.\nConsequently, the values shown (~0.01–0.02) represent the relative abundance of phage\nΦV-1 DNA per host-cell equivalent (e.g., 0.02 indicates 0.02 phage genomes per bacterial\ngenome). The phage RD is naturally below 1 because it reflects its absolute abundance\nrelative to this internal baseline, not a fold-change relative to an uninfected control. The\nupdated legend for Fig. 6a now explicitly states: “The RD was normalized to the sample\nspecific genomic average RD of the combined E. coli–ΦV-1 reference genome.”\nWe have revised the Methods as follows:\n“Sample Preparation for DNA-seq data (WGS data)\nE. coli MG1655 cultures harboring the indicated plasmids were grown in LB medium\nsupplemented with 0.2% (w/v) L‑arabinose to an OD600 of 0.5 and then infected with\nEscherichia phage ΦV‑1 at a MOI of 3. After 5 or 20 min of infection, cultures were rapidly\nchilled on ice, harvested by centrifugation at 4 °C, and stored at ‑80 °C. Total genomic DNA\nwas extracted using the Bacterial Genomic DNA Extraction Kit (DP302, TIANGEN). DNA\nconcentration and purity were measured with a NanoDrop 2000 spectrophotometer.\nLibrary Construction and Sequencing\nFor DNA sequencing, genomic DNA (200 ng) was fragmented to ~300–350 bp using a Covaris\nultrasonicator. Fragmented DNA was end‑repaired, 5′‑phosphorylated, 3′‑A‑tailed, and\nligated to sequencing adaptors. Libraries were size‑selected using DNA cleanup beads and\namplified by PCR for eight cycles. Library quality was assessed on an Agilent 2100 Bioanalyzer.\nQualified libraries were sequenced in paired‑end mode (PE150) on Illumina platforms\n(Novaseq6000).\nBioinformatic Processing\nRaw sequencing reads were processed with fastp (v0.23.0) to remove adaptor sequences, PCR\nprimers, reads containing >14 ambiguous bases (N), and low‑quality reads with >40% of\nbases below Q20. Clean reads were aligned to a combined reference genome consisting of\nthe E. coli MG1655 chromosome (GenBank accession: U00096.3) and the complete phage\nΦV‑1 genome (NC_001426.1) using the Sentieon pipeline (v202112.02). Duplicate reads were\nremoved, and single‑nucleotide variants (SNVs) and insertions/deletions (InDels) were called\nusing Sentieon’s variant‑calling modules.\nRead‑Depth (RD) Analysis and Normalization\nTo determine the relative abundance of phage DNA, we performed read‑depth (RD) analysis\nfollowing principles established for copy‑number variation (CNV) detection [34]. The analysis\nwas carried out with CNVnator (v0.3.1) using a bin size of 100 bp. A sample‑specific genomic\nbaseline was established by analyzing the global distribution of RD across the entire\ncombined reference genome. The RD for each genomic segment was then normalized against\nthis internal baseline. Phage genome abundance was quantified as the mean normalized RD\nacross all bins spanning the combined reference genome. This value represents the relative\ncopy number of phage DNA per host‑genome equivalent, where a value of 1.0 corresponds to\none phage genome per bacterial genome. The normalized RD values reflect the relative ratio\nof phage‑to‑host DNA in each sample.”\nWe hope these clarifications and methodological additions fully address the reviewer’s\nconcerns. We are grateful for the reviewer’s careful attention, which has enabled us to\nprovide a more complete and transparent description of our analytical approach.\n- The authors now present a model wherein Upx functions as an abortive-infection system.\nThis would imply that gp16 triggers Upx-mediated toxicity in the absence of phage infection.\nThey have included data showing that gp16 itself is not toxic, but these data do not address\nthe question of whether gp16 is an in vivo trigger of Upx-mediated toxicity (e.g. abortive\ninfection). My previous review was poorly worded, and I apologize for this, but the question\nat hand is whether gp16 activates Upx and the new data do not address this. However, the\nnew data included in Figure 7C showing a growth curve of cells expressing Upx infected at\ndifferent MOIs shows a phenotype similar to those for direct defense systems in the\nliterature, not an abortive system in my view. I suggest the authors review Vasallo et al., 2022\nFigure 3B and Extended Data Figure 3 and other manuscripts on direct defense systems. If\nthe authors agree that Upx is performing like a direct system, I would suggest revising the\ntext. The gp16 toxicity data by themselves are unfortunately not that meaningful. They could\neither be removed, or replaced with an experiment testing whether gp16 triggers\nUpx-mediated toxicity in a two-plasmid system. If it does not, this would further agree with\nthe concept that Upx is a direct system wherein gp16 may play a role in facilitating activity in\nvivo. However, I don’t think this toxicity experiment is essential in light of the MOI curve in\n7C.\nResponse: We sincerely thank the reviewer for their exceptionally clear and helpful guidance\non interpreting the functional class of Upx. We fully agree with the reviewer’s assessment.\nUpon re-evaluating our data—specifically, the robust growth recovery of E. coli expressing\nUpx following ΦV-1 infection at both low (MOI = 0.1) and high (MOI = 3) multiplicities—we\nfind that this phenotype aligns with the paradigms established for direct defense systems in\nthe suggested literature (Vasallo et al., 2022). We therefore concur that Upx operates as a\ndirect defense system rather than an abortive-infection system.\nAccordingly, we have implemented the following revisions in the manuscript:\n1. Text updated throughout: We have revised the Results section to consistently describe Upx\nas a direct defense system. All references to \"abortive-infection-like\" mechanisms have been\nreplaced.\n2. Figure panel removed: In line with the reviewer’s suggestion that the gp16 toxicity data by\nthemselves are unfortunately not that meaningful, we have removed the corresponding\nnon-toxicity data panel (formerly Fig. S6g).\nWe are grateful for this critical insight, which has significantly sharpened the conceptual\nfocus of our work."}, {"round": 3, "reviewer_comments": {"reviewer_3": "All of my comments regarding this manuscript have now been addressed, I am excited to see it published in Nature Communications. I thank the authors for their responses, and am glad they found my comments helpful."}, "author_response": ""}]

Decision letter