Structural analysis of the BisI family of modification dependent restriction endonucleases.

The BisI family of restriction endonucleases is unique in requiring multiple methylated or hydroxymethylated cytosine residues within a short recognition sequence (GCNGC), and in cleaving directly within this sequence, rather than at a distance. Here, we report that the number of modified cytosines that are required for cleavage can be tuned by the salt concentration. We present crystal structures of two members of the BisI family, NhoI and Eco15I_Ntd (N-terminal domain of Eco15I), in the absence of DNA and in specific complexes with tetra-methylated GCNGC target DNA. The structures show that NhoI and Eco15I_Ntd sense modified cytosine bases in the context of double-stranded DNA (dsDNA) without base flipping. In the co-crystal structures of NhoI and Eco15I_Ntd with DNA, the internal methyl groups (G5mCNGC) interact with the side chains of an (H/R)(V/I/T/M) di-amino acid motif near the C-terminus of the distal enzyme subunit and arginine residue from the proximal subunit. The external methyl groups (GCNG5mC) interact with the proximal enzyme subunit, mostly through main chain contacts. Surface plasmon resonance analysis for Eco15I_Ntd shows that the internal and external methyl binding pockets contribute about equally to sensing of cytosine methyl groups.

Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI.

Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.

Fusion of FokI and catalytically inactive prokaryotic Argonautes enables site-specific programmable DNA cleavage.

Site-specific nucleases are crucial for genome engineering applications in medicine and agriculture. The ideal site-specific nucleases are easily reprogrammable, highly specific in target site recognition, and robust in nuclease activities. Prokaryotic Argonaute (pAgo) proteins have received much attention as biotechnological tools due to their ability to recognize specific target sequences without a protospacer adjacent motif, but their lack of intrinsic dsDNA unwinding activity limits their utility in key applications such as gene editing. Recently, we developed a pAgo-based system for site-specific DNA cleavage at physiological temperatures independently of the DNA form, using peptide nucleic acids (PNAs) to facilitate unwinding dsDNA targets. Here, we fused catalytically dead pAgos with the nuclease domain of the restriction endonuclease FokI and named this modified platform PNA-assisted FokI-(d)pAgo (PNFP) editors. In the PNFP system, catalytically inactive pAgo recognizes and binds to a specific target DNA sequence based on a programmable guide DNA sequence; upon binding to the target site, the FokI domains dimerize and introduce precise dsDNA breaks. We explored key parameters of the PNFP system including the requirements of PNA and guide DNAs, the specificity of PNA and guide DNA on target cleavage, the optimal concentration of different components, reaction time for invasion and cleavage, and ideal temperature and reaction buffer, to ensure efficient DNA editing in vitro. The results demonstrated robust site-specific target cleavage by PNFP system at optimal conditions in vitro. We envision that the PNFP system will provide higher editing efficiency and specificity with fewer off-target effects in vivo.

Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.

Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.

Orientation-Guided Immobilization of Probe DNA on swCNT-FET for Enhancing Sensitivity of EcoRV Detection.

We present a novel approach that integrates electrical measurements with molecular dynamics (MD) simulations to assess the activity of type-II restriction endonucleases, specifically EcoRV. Our approach employs a single-walled carbon nanotube field-effect transistor (swCNT-FET) functionalized with the EcoRV substrate DNA, enabling the detection of enzymatic cleavage events. Notably, we leveraged the methylene blue (MB) tag as an "orientation guide" to immobilize the EcoRV substrate DNA in a specific direction, thereby enhancing the proximity of the DNA cleavage reaction to the swCNT surface and consequently improving the sensitivity in EcoRV detection. We conducted computational modeling to compare the conformations and electrostatic potential (ESP) of MB-tagged DNA with its MB-free counterpart, providing strong support for our electrical measurements. Both conformational and ESP simulations exhibited robust agreement with our experimental data. The inhibitory efficacy of the EcoRV inhibitor aurintricarboxylic acid (ATA) was also evaluated, and the selectivity of the sensing device was examined.

DNA Sequence Control of Enzyme Filamentation and Activation of the SgrAI Endonuclease.

Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

A Life Investigating Pathways That Repair Broken Chromosomes.

Double-strand breaks (DSBs) pose a severe challenge to genome integrity; consequently, cells have developed efficient mechanisms to repair DSBs through several pathways of homologous recombination and other nonhomologous end-joining processes. Much of our understanding of these pathways has come from the analysis of site-specific DSBs created by the HO endonuclease in the budding yeast Saccharomyces cerevisiae. I was fortunate to get in on the ground floor of analyzing the fate of synchronously induced DSBs through the study of what I coined "in vivo biochemistry." I have had the remarkable good fortune to profit from the development of new techniques that have permitted an ever more detailed dissection of these repair mechanisms, which are described here.

High-throughput direct screening of restriction endonuclease using a microfluidic fluorescence-activated drop sorter based on the SOS response in Escherichia coli.

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route to expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient manner. We constructed a host bacterial cell to link the genotype of REs to the phenotype of ß-galactosidase expression based on the bacterial SOS response, and used a high-throughput microfluidic platform to isolate, detect and sort the REs in microfluidic drops at a frequency of ∼800 drops per second. We employed this strategy to screen for the XbaI gene from the constructed libraries of varied sizes. In a single round of sorting, a 90-fold target enrichment was achieved within 1 h. Compared to conventional RE-screening methods, the direct screening approach that we propose excels at efficient search of desirable REs in natural samples - especially unculturable samples - and can be tailored to high-throughput screening of a wide range of genotoxic targets.

Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in ß-thalassemia and sickle cell disease patients.

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both ß-thalassemia (ß-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.e. Gg promoters) were knocked into the Gg gene(s) of K562 cells via CRISPR/Cas9. Subsequently, the GFP fluorescence levels were compared in the ensuing cell populations and isolated clones. In both instances, median fluorescence intensities (MFI) of the knockin cells having the inserted XmnI T allele were higher than those having the XmnI C allele. Our results suggest that the XmnI T allele can increase Gg expression in K562 cells. The possible functional significance of the XmnI Gg -158 C/T polymorphism provides a rationale for the aforementioned associations. Furthermore, the XmnI polymorphism as a functional SNP substantiates its importance as a prognostic marker.

The Cohesin Complex Prevents the End Joining of Distant DNA Double-Strand Ends.

The end joining of distant DNA double-strand ends (DSEs) can produce potentially deleterious rearrangements. We show that depletion of cohesion complex proteins specifically stimulates the end joining (both C-NHEJ and A-EJ) of distant, but not close, I-SceI-induced DSEs in S/G2 phases. At the genome level, whole-exome sequencing showed that ablation of RAD21 or Sororin produces large chromosomal rearrangements (translocation, duplication, deletion). Moreover, cytogenetic analysis showed that RAD21 silencing leads to the formation of chromosome fusions synergistically with replication stress, which generates distant single-ended DSEs. These data reveal a role for the cohesin complex in protecting against genome rearrangements arising from the ligation of distant DSEs in S/G2 phases (both long-range DSEs and those that are only a few kilobases apart), while keeping end joining fully active for close DSEs. Therefore, this role likely involves limitation of DSE motility specifically in S phase, rather than inhibition of the end-joining machinery itself.

Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity.

Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes-the acceleration of DNA cleavage and the expansion of sequence specificity-are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca2+ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme-DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein-DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.

The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

Ash1 and Tup1 dependent repression of the Saccharomyces cerevisiae HO promoter requires activator-dependent nucleosome eviction.

Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.

A single Ho-induced double-strand break at the MAT locus is lethal in Candida glabrata.

Mating-type switching is a complex mechanism that promotes sexual reproduction in Saccharomycotina. In the model species Saccharomyces cerevisiae, mating-type switching is initiated by the Ho endonuclease that performs a site-specific double-strand break (DSB) at MAT, repaired by homologous recombination (HR) using one of the two silent mating-type loci, HMLalpha and HMRa. The reasons why all the elements of the mating-type switching system have been conserved in some Saccharomycotina, that do not show a sexual cycle nor mating-type switching, remain unknown. To gain insight on this phenomenon, we used the yeast Candida glabrata, phylogenetically close to S. cerevisiae, and for which no spontaneous and efficient mating-type switching has been observed. We have previously shown that expression of S. cerevisiae's Ho (ScHo) gene triggers mating-type switching in C. glabrata, but this leads to massive cell death. In addition, we unexpectedly found, that not only MAT but also HML was cut in this species, suggesting the formation of multiple chromosomal DSBs upon HO induction. We now report that HMR is also cut by ScHo in wild-type strains of C. glabrata. To understand the link between mating-type switching and cell death in C. glabrata, we constructed strains mutated precisely at the Ho recognition sites. We find that even when HML and HMR are protected from the Ho-cut, introducing a DSB at MAT is sufficient to induce cell death, whereas one DSB at HML or HMR is not. We demonstrate that mating-type switching in C. glabrata can be triggered using CRISPR-Cas9, without high lethality. We also show that switching is Rad51-dependent, as in S. cerevisiae, but that donor preference is not conserved in C. glabrata. Altogether, these results suggest that a DSB at MAT can be repaired by HR in C. glabrata, but that repair is prevented by ScHo.

A Rad51-independent pathway promotes single-strand template repair in gene editing.

The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.

Assembly of Synaptic Protein-DNA Complexes: Critical Role of Non-Specific Interactions.

The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.

Quantum biology.π-πentanglement signatures in protein-DNA interactions.

The biological functions of DNA are carried out by individual proteins that interact with specific sequences along the DNA in order to prime the molecular processes required by the cellular metabolism. Protein-DNA interactions include DNA replication, gene expression and its regulation, DNA repair, DNA restriction and modification by endonucleases, generally classified as enzymatic functions, or transcription factors functions. To find specific binding target sequences and achieve their aims, in less than one second proteins operate in symbiosis with a crowded cellular environment, identifying extremely small cognate sequences along the DNA chain, which range from 15-20 bps for repressors to 4-6 bps for restriction enzymes. In a previous work, we proposed that the extraordinary ability of proteins to identify consensus sequences on DNA in a short time appears to be dependent on specific quantum signatures such as the entanglement ofπ-πelectrons between DNA nucleotides and protein amino acids, where the couple ofπelectrons function as a radical pair, oneπelectron is located on a specific site of sequence to be identified and the other one performs a quantum walk to identify possible sites of consensus sequence. In this paper, we use the restriction endonucleases enzymes, EcoRV and EcoRI as a case study. These enzymes are able to recognize 3'-GATACT-5' or 3'-GAATCT-5' sequences, respectively. We exploit the analogy of a coin operator with a Bloch sphere to demonstrate that the entanglement betweenπ-πelectrons generated at the contacts on specific GA dimers between proteins and DNA relies on the spin of the electrons that form an initial singlet state. The latter is a maximally entangled state so that the identification of specific nucleotides is associated with the formation of singlet states. On the other hand, during the identification of subsequent GA dimers, the spin-orbit interaction on walkingπelectron induces triplet transitions so that singlet-triplet transitions should manifest an experimentally measurable effect. We propose that the possible experimental evidence of entanglement betweenπ-πelectrons may be due to the phosphorescence signal correspondence to triplet decay processes.

I-SceI and customized meganucleases-mediated genome editing in tomato and oilseed rape.

Meganucleases are rare cutting enzymes that can generate DNA modifications and are part of the plant genome editing toolkit although they lack versatility. Here, we evaluated the use of two meganucleases, I-SceI and a customized meganuclease, in tomato and oilseed rape. Different strategies were explored for the use of these meganucleases. The activity of a customized and a I-SceI meganucleases was first estimated by the use of a reporter construct GFFP with the target sequences and enabled to demonstrate that both meganucleases can generate double-strand break and HDR mediated recombination in a reporter gene. Interestingly, I-SceI seems to have a higher DSB efficiency than the customized meganuclease: up to 62.5% in tomato and 44.8% in oilseed rape. Secondly, the same exogenous landing pad was introduced in both species. Despite being less efficient compared to I-SceI, the customized meganuclease was able to generate the excision of an exogenous transgene (large deletion of up to 3316 bp) present in tomato. In this paper, we also present some pitfalls to be considered before using meganucleases (e.g., potential toxicity) for plant genome editing.

FACT and Ash1 promote long-range and bidirectional nucleosome eviction at the HO promoter.

The Saccharomyces cerevisiae HO gene is a model regulatory system with complex transcriptional regulation. Budding yeast divide asymmetrically and HO is expressed only in mother cells where a nucleosome eviction cascade along the promoter during the cell cycle enables activation. HO expression in daughter cells is inhibited by high concentration of Ash1 in daughters. To understand how Ash1 represses transcription, we used a myo4 mutation which boosts Ash1 accumulation in both mothers and daughters and show that Ash1 inhibits promoter recruitment of SWI/SNF and Gcn5. We show Ash1 is also required for the efficient nucleosome repopulation that occurs after eviction, and the strongest effects of Ash1 are seen when Ash1 has been degraded and at promoter locations distant from where Ash1 bound. Additionally, we defined a specific nucleosome/nucleosome-depleted region structure that restricts HO activation to one of two paralogous DNA-binding factors. We also show that nucleosome eviction occurs bidirectionally over a large distance. Significantly, eviction of the more distant nucleosomes is dependent upon the FACT histone chaperone, and FACT is recruited to these regions when eviction is beginning. These last observations, along with ChIP experiments involving the SBF factor, suggest a long-distance loop transiently forms at the HO promoter.

Structure of HhaI endonuclease with cognate DNA at an atomic resolution of 1.0 Å.

HhaI, a Type II restriction endonuclease, recognizes the symmetric sequence 5'-GCG↓C-3' in duplex DNA and cleaves ('↓') to produce fragments with 2-base, 3'-overhangs. We determined the structure of HhaI in complex with cognate DNA at an ultra-high atomic resolution of 1.0 Å. Most restriction enzymes act as dimers with two catalytic sites, and cleave the two strands of duplex DNA simultaneously, in a single binding event. HhaI, in contrast, acts as a monomer with only one catalytic site, and cleaves the DNA strands sequentially, one after the other. HhaI comprises three domains, each consisting of a mixed five-stranded ß sheet with a defined function. The first domain contains the catalytic-site; the second contains residues for sequence recognition; and the third contributes to non-specific DNA binding. The active-site belongs to the 'PD-D/EXK' superfamily of nucleases and contains the motif SD-X11-EAK. The first two domains are similar in structure to two other monomeric restriction enzymes, HinP1I (G↓CGC) and MspI (C↓CGG), which produce fragments with 5'-overhangs. The third domain, present only in HhaI, shifts the positions of the recognition residues relative to the catalytic site enabling this enzyme to cleave the recognition sequence at a different position. The structure of M.HhaI, the biological methyltransferase partner of HhaI, was determined earlier. Together, these two structures represent the first natural pair of restriction-modification enzymes to be characterized in atomic detail.