Modification-dependent Restriction Endonuclease Research Articles

The Restriction Activity Investigation of Rv2528c, an Mrr-like Modification-Dependent Restriction Endonuclease from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), as a typical intracellular pathogen, possesses several putative restriction-modification (R-M) systems, which restrict exogenous DNA's entry, such as bacterial phage infection. Here, we investigate Rv2528c, a putative Mrr-like type IV restriction endonuclease (REase) from Mtb H37Rv, which is predicted to degrade methylated DNA that contains m6A, m5C, etc. Rv2528c shows significant cytotoxicity after being expressed in Escherichia coli BL21(DE3)pLysS strain. The Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay indicates that Rv2528c cleaves genomic DNA in vivo. The plasmid transformation efficiency of BL21(DE3)pLysS strain harboring Rv2528c gene was obviously decreased after plasmids were in vitro methylated by commercial DNA methyltransferases such as M.EcoGII, M.HhaI, etc. These results are consistent with the characteristics of type IV REases. The in vitro DNA cleavage condition and the consensus cleavage/recognition site of Rv2528c still remain unclear, similar to that of most Mrr-family proteins. The possible reasons mentioned above and the potential role of Rv2528c for Mtb were discussed.

Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems.

Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and HhiV4I act as sensors of adenine methylation in the 6mApT (N6-methyladenine, 6mA, or N6mA) context. DNA-binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK-wH (FcyTI, Psp4BI), PUA-wH-HNH (HtuIII), wH-GIY-YIG (Ahi29725I, Apa233I), and PLD-wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam+ Gm6ATC sequence contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD-wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other modification-dependent restriction endonucleases (MDREs, also called type IV restriction systems), the new fusion endonucleases except those in the PD-(D/E)XK-wH family cleave close to but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation readers. Other potential 6mA sensors in modified DNA are also discussed.

A Unique m6A-Dependent Restriction Endonuclease from an Archaeal Virus

Prokaryotes possess numerous diverse defense systems to resist viral infections, while some viruses have also evolved antiviral defense systems to exclude other viruses in cases of multiple infections. Here, we report the first virus-derived modification-dependent restriction endonuclease (HHPV4I) from the archaeal virus HHPV4 (Haloarcula hispanica pleomorphic virus 4). HHPV4I contains an SRA domain, a winged helix (wH) domain, and an HNH domain; recognizes the Gm6ATC site; and specifically binds to Gm6ATC site-containing DNA. Both the wH domain and the HNH domain are responsible for DNA binding. Unlike the well-known m6A-specific restriction enzyme DpnI, HHPV4I only efficiently cleaves DNA with a fully methylated Gm6ATC site and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. Furthermore, HHPV4I preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC site. Thus, the cleavage pattern of HHPV4I is distinct from those of all of the presently characterized restriction endonucleases. Mutations in the wH domain of HHPV4I do not alter m6A-dependent endonuclease activity, but they decrease recognition sequence specificity, thus expanding the cleaving capacity to more m6A-containing DNA sequences. The wH domain provides a target for searching, developing, and engineering novel m6A-dependent endonucleases. IMPORTANCE Many modification-dependent restriction endonucleases (MDREs) were identified in prokaryotes and recognized modified cytosine bases, such as 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), and glucosyl-5-hydroxymethylcytosine (g5hmC). The first virus-derived MDRE (HHPV4I) from the archaeal virus HHPV4 was identified in this study. The viral MDRE suggested a new strategy employed by the virus to exclude other viruses in the case of multiple replications. HHPV4I is a novel N6-methyladenine (m6A)-dependent restriction endonuclease, while the cleavage pattern of HHPV4I is distinct from the well-known m6A-dependent restriction endonuclease DpnI. HHPV4I recognizes Gm6ATC sites and cleaves DNA both upstream and downstream of the Gm6ATC sites on both DNA strands. It preferentially cleaves DNA between VR bases (V = A/G/C, R = A/G) 4 to 20 nt away from the Gm6ATC sites. Furthermore, mutations in the HHPV4I wH domain can alter the sequence specificity without impeding the m6A-dependent DNA cleavage activity, providing a target for engineering more m6A-dependent endonucleases with different sequence specificities.

High-throughput sequencing of EcoWI restriction fragments maps the genome-wide landscape of phosphorothioate modification at base resolution.

Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genomes and their exact roles in the cell remain to be defined. In this study, we developed a simple and convenient technique called EcoWI-seq based on a modification-dependent restriction endonuclease to identify genomic positions of PT modifications. EcoWI-seq shows similar performance than other PT modification detection techniques and additionally, is easily scalable while requiring little starting material. As a proof of principle, we applied EcoWI-seq to map the PT modifications at base resolution in the genomes of both the Salmonella enterica cerro 87 and E. coli expressing the dnd+ gene cluster. Specifically, we address whether the partial establishment of modified PT positions is a stochastic or deterministic process. EcoWI-seq reveals a systematic usage of the same subset of target sites in clones for which the PT modification has been independently established.

Crystal structures of the EVE-HNH endonuclease VcaM4I in the presence and absence of DNA.

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.

Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine.

Aba-Seq (DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing) provides a cost-effective and non-chemical based method for the high-resolution mapping of genomic 5-hydroxymethylcytosine (5hmC). The high specificity of the AbaSI enzyme allows sensitive detection of 5hmC in the genome. Here, we describe the Aba-Seq technique that was used for the high-resolution mapping of 5hmC in the genome of mouse embryonic stem cells (E14).

Protein Domain Guided Screen for Sequence Specific and Phosphorothioate-Dependent Restriction Endonucleases.

Modification dependent restriction endonucleases (MDREs) restrict modified DNA, typically with limited sequence specificity (∼2–4 bp). Here, we focus on MDREs that have an SRA and/or SBD (sulfur binding domain) fused to an HNH endonuclease domain, cleaving cytosine modified or phosphorothioated (PT) DNA. We independently characterized the SBD-SRA-HNH endonuclease ScoMcrA, which preferentially cleaves 5hmC modified DNA. We report five SBD-HNH endonucleases, all recognizing GpsAAC/GpsTTC sequence and cleaving outside with a single nucleotide 3′ stagger: EcoWI (N7/N6), Ksp11411I (N5/N4), Bsp305I (N6/N4-5), Mae9806I [N(8-10)/N(8-9)], and Sau43800I [N(8-9)/N(7-8)]. EcoWI and Bsp305I are more specific for PT modified DNA in Mg2+ buffer, and promiscuous with Mn2+. Ksp11411I is more PT specific with Ni2+. EcoWI and Ksp11411I cleave fully- and hemi-PT modified oligos, while Bsp305I cleaves only fully modified ones. EcoWI forms a dimer in solution and cleaves more efficiently in the presence of two modified sites. In addition, we demonstrate that EcoWI PT-dependent activity has biological function: EcoWI expressing cells restrict dnd+ GpsAAC modified plasmid strongly, and GpsGCC DNA weakly. This work establishes a framework for biotechnology applications of PT-dependent restriction endonucleases (PTDRs).

Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples

BackgroundOwing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined.MethodsDNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA.ResultsMspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI + sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration + sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples.ConclusionThis optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.

Crystal structure of the modification-dependent SRA-HNH endonuclease TagI.

TagI belongs to the recently characterized SRA-HNH family of modification-dependent restriction endonucleases (REases) that also includes ScoA3IV (Sco5333) and TbiR51I (Tbis1). Here, we present a crystal structure of dimeric TagI, which exhibits a DNA binding site formed jointly by the nuclease domains, and separate binding sites for modified DNA bases in the two protomers. The nuclease domains have characteristic features of HNH/ββα-Me REases, and catalyze nicks or double strand breaks, with preference for /RY and RYN/RY sites, respectively. The SRA domains have the canonical fold. Their pockets for the flipped bases are spacious enough to accommodate 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC), but not glucosyl-5-hydroxymethylcytosine (g5hmC). Such preference is in agreement with the biochemical determination of the TagI modification dependence and the results of phage restriction assays. The ability of TagI to digest plasmids methylated by Dcm (C5mCWGG), M.Fnu4HI (G5mCNGC) or M.HpyCH4IV (A5mCGT) suggests that the SRA domains of the enzyme are tolerant to different sequence contexts of the modified base.

Phylogenomics and sequence-structure-function relationships in the GmrSD family of Type IV restriction enzymes.

BackgroundGmrSD is a modification-dependent restriction endonuclease that specifically targets and cleaves glucosylated hydroxymethylcytosine (glc-HMC) modified DNA. It is encoded either as two separate single-domain GmrS and GmrD proteins or as a single protein carrying both domains. Previous studies suggested that GmrS acts as endonuclease and NTPase whereas GmrD binds DNA.MethodsIn this work we applied homology detection, sequence conservation analysis, fold recognition and homology modeling methods to study sequence-structure-function relationships in the GmrSD restriction endonucleases family. We also analyzed the phylogeny and genomic context of the family members.ResultsResults of our comparative genomics study show that GmrS exhibits similarity to proteins from the ParB/Srx fold which can have both NTPase and nuclease activity. In contrast to the previous studies though, we attribute the nuclease activity also to GmrD as we found it to contain the HNH endonuclease motif. We revealed residues potentially important for structure and function in both domains. Moreover, we found that GmrSD systems exist predominantly as a fused, double-domain form rather than as a heterodimer and that their homologs are often encoded in regions enriched in defense and gene mobility-related elements. Finally, phylogenetic reconstructions of GmrS and GmrD domains revealed that they coevolved and only few GmrSD systems appear to be assembled from distantly related GmrS and GmrD components.ConclusionsOur study provides insight into sequence-structure-function relationships in the yet poorly characterized family of Type IV restriction enzymes. Comparative genomics allowed to propose possible role of GmrD domain in the function of the GmrSD enzyme and possible active sites of both GmrS and GmrD domains. Presented results can guide further experimental characterization of these enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0773-z) contains supplementary material, which is available to authorized users.

Structure-guided sequence specificity engineering of the modification-dependent restriction endonuclease LpnPI.

The eukaryotic Set and Ring Associated (SRA) domains and structurally similar DNA recognition domains of prokaryotic cytosine modification-dependent restriction endonucleases recognize methylated, hydroxymethylated or glucosylated cytosine in various sequence contexts. Here, we report the apo-structure of the N-terminal SRA-like domain of the cytosine modification-dependent restriction enzyme LpnPI that recognizes modified cytosine in the 5′-C(mC)DG-3′ target sequence (where mC is 5-methylcytosine or 5-hydroxymethylcytosine and D = A/T/G). Structure-guided mutational analysis revealed LpnPI residues involved in base-specific interactions and demonstrated binding site plasticity that allowed limited target sequence degeneracy. Furthermore, modular exchange of the LpnPI specificity loops by structural equivalents of related enzymes AspBHI and SgrTI altered sequence specificity of LpnPI. Taken together, our results pave the way for specificity engineering of the cytosine modification-dependent restriction enzymes.

Crystallization and preliminary X-ray analysis of the type IV restriction endonuclease ScoMcrA from Streptomyces coelicolor, which cleaves both Dcm-methylated DNA and phosphorothioated DNA.

ScoMcrA is a type IV modification-dependent restriction endonuclease found in the model strain Streptomyces coelicolor. Unlike type I, II and III restriction endonucleases, which cleave unmodified DNA, type IV restriction endonucleases cleave modified DNA, including methylated, hydroxymethylated, glucosyl-hydroxymethylated and phosphorothioated DNA. ScoMcrA targets both Dcm-methylated DNA and phosphorothioated DNA, and makes double-strand breaks 16-28 nt away from the modified nucleotides or the phosphorothioate links. However, the mechanism by which ScoMcrA recognizes these two entirely different types of modification remains unclear. In this study, the ScoMcrA protein was overexpressed, purified and crystallized. The crystals diffracted to 3.35 Å resolution and belonged to space group P2(1)2(1)2(1). The unit-cell parameters were determined to be a=130.19, b=139.36, c=281.01 Å, α=β=γ=90°. These results will facilitate the detailed structural analysis of ScoMcrA and further elucidation of its biochemical mechanism.

Chemical display of pyrimidine bases flipped out by modification-dependent restriction endonucleases of MspJI and PvuRts1I families.

The epigenetic DNA modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in eukaryotes are recognized either in the context of double-stranded DNA (e.g., by the methyl-CpG binding domain of MeCP2), or in the flipped-out state (e.g., by the SRA domain of UHRF1). The SRA-like domains and the base-flipping mechanism for 5(h)mC recognition are also shared by the recently discovered prokaryotic modification-dependent endonucleases of the MspJI and PvuRts1I families. Since the mechanism of modified cytosine recognition by many potential eukaryotic and prokaryotic 5(h)mC “readers” is still unknown, a fast solution based method for the detection of extrahelical 5(h)mC would be very useful. In the present study we tested base-flipping by MspJI- and PvuRts1I-like restriction enzymes using several solution-based methods, including fluorescence measurements of the cytosine analog pyrrolocytosine and chemical modification of extrahelical pyrimidines with chloroacetaldehyde and KMnO4. We find that only KMnO4 proved an efficient probe for the positive display of flipped out pyrimidines, albeit the method required either non-physiological pH (4.3) or a substitution of the target cytosine with thymine. Our results imply that DNA recognition mechanism of 5(h)mC binding proteins should be tested using a combination of all available methods, as the lack of a positive signal in some assays does not exclude the base flipping mechanism.

Modification-dependent restriction endonuclease, MspJI, flips 5-methylcytosine out of the DNA helix.

MspJI belongs to a family of restriction enzymes that cleave DNA containing 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC). MspJI is specific for the sequence 5(h)mC-N-N-G or A and cleaves with some variability 9/13 nucleotides downstream. Earlier, we reported the crystal structure of MspJI without DNA and proposed how it might recognize this sequence and catalyze cleavage. Here we report its co-crystal structure with a 27-base pair oligonucleotide containing 5mC. This structure confirms that MspJI acts as a homotetramer and that the modified cytosine is flipped from the DNA helix into an SRA-like-binding pocket. We expected the structure to reveal two DNA molecules bound specifically to the tetramer and engaged with the enzyme's two DNA-cleavage sites. A coincidence of crystal packing precluded this organization, however. We found that each DNA molecule interacted with two adjacent tetramers, binding one specifically and the other non-specifically. The latter interaction, which prevented cleavage-site engagement, also involved base flipping and might represent the sequence-interrogation phase that precedes specific recognition. MspJI is unusual in that DNA molecules are recognized and cleaved by different subunits. Such interchange of function might explain how other complex multimeric restriction enzymes act.

Structure of 5-hydroxymethylcytosine-specific restriction enzyme, AbaSI, in complex with DNA.

AbaSI, a member of the PvuRts1I-family of modification-dependent restriction endonucleases, cleaves deoxyribonucleic acid (DNA) containing 5-hydroxymethylctosine (5hmC) and glucosylated 5hmC (g5hmC), but not DNA containing unmodified cytosine. AbaSI has been used as a tool for mapping the genomic locations of 5hmC, an important epigenetic modification in the DNA of higher organisms. Here we report the crystal structures of AbaSI in the presence and absence of DNA. These structures provide considerable, although incomplete, insight into how this enzyme acts. AbaSI appears to be mainly a homodimer in solution, but interacts with DNA in our structures as a homotetramer. Each AbaSI subunit comprises an N-terminal, Vsr-like, cleavage domain containing a single catalytic site, and a C-terminal, SRA-like, 5hmC-binding domain. Two N-terminal helices mediate most of the homodimer interface. Dimerization brings together the two catalytic sites required for double-strand cleavage, and separates the 5hmC binding-domains by ∼70 Å, consistent with the known activity of AbaSI which cleaves DNA optimally between symmetrically modified cytosines ∼22 bp apart. The eukaryotic SET and RING-associated (SRA) domains bind to DNA containing 5-methylcytosine (5mC) in the hemi-methylated CpG sequence. They make contacts in both the major and minor DNA grooves, and flip the modified cytosine out of the helix into a conserved binding pocket. In contrast, the SRA-like domain of AbaSI, which has no sequence specificity, contacts only the minor DNA groove, and in our current structures the 5hmC remains intra-helical. A conserved, binding pocket is nevertheless present in this domain, suitable for accommodating 5hmC and g5hmC. We consider it likely, therefore, that base-flipping is part of the recognition and cleavage mechanism of AbaSI, but that our structures represent an earlier, pre-flipped stage, prior to actual recognition.

Structure and mutagenesis of the DNA modification-dependent restriction endonuclease AspBHI

The modification-dependent restriction endonuclease AspBHI recognizes 5-methylcytosine (5mC) in the double-strand DNA sequence context of (C/T)(C/G)(5mC)N(C/G) (N = any nucleotide) and cleaves the two strands a fixed distance (N12/N16) 3′ to the modified cytosine. We determined the crystal structure of the homo-tetrameric AspBHI. Each subunit of the protein comprises two domains: an N-terminal DNA-recognition domain and a C-terminal DNA cleavage domain. The N-terminal domain is structurally similar to the eukaryotic SET and RING-associated (SRA) domain, which is known to bind to a hemi-methylated CpG dinucleotide. The C-terminal domain is structurally similar to classic Type II restriction enzymes and contains the endonuclease catalytic-site motif of DX20EAK. To understand how specific amino acids affect AspBHI recognition preference, we generated a homology model of the AspBHI-DNA complex, and probed the importance of individual amino acids by mutagenesis. Ser41 and Arg42 are predicted to be located in the DNA minor groove 5′ to the modified cytosine. Substitution of Ser41 with alanine (S41A) and cysteine (S41C) resulted in mutants with altered cleavage activity. All 19 Arg42 variants resulted in loss of endonuclease activity.

High-throughput sequencing of methylated cytosine enriched by modification-dependent restriction endonuclease MspJI

BackgroundAs a well-known epigenomic modification, DNA methylation is found to be common in plants and plays an important role in many biological processes. Relying on the unique feature of methylation-dependent digestion, the family of methylation-requiring restriction-like endonuclease, such as MspJI and its homologs, was suggested for a potential usage in methylation detection.ResultsIn this study, we combine MspJI digestion and electrophoretic band selection with next generation high-throughput sequencing technology to detect 5-methylcytosines in Arabidopsis genome. By developing a bioinformatics workflow to attribute the CNNR sites recognized by MspJI to the reference genome, we fulfilled the systematic assessment of this method.ConclusionsAccording to the assessment, here we provide the method for generating a detailed map of plant methylome that could be feasible, reliable and economical in methylation investigation.

Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease

The MspJI modification-dependent restriction endonuclease recognizes 5-methylcytosine or 5-hydroxymethylcytosine in the context of CNN(G/A) and cleaves both strands at fixed distances (N12/N16) away from the modified cytosine at the 3′-side. We determined the crystal structure of MspJI of Mycobacterium sp. JLS at 2.05-Å resolution. Each protein monomer harbors two domains: an N-terminal DNA-binding domain and a C-terminal endonuclease. The N-terminal domain is structurally similar to that of the eukaryotic SET and RING-associated domain, which is known to bind to a hemi-methylated CpG dinucleotide. Four protein monomers are found in the crystallographic asymmetric unit. Analytical gel-filtration and ultracentrifugation measurements confirm that the protein exists as a tetramer in solution. Two monomers form a back-to-back dimer mediated by their C-terminal endonuclease domains. Two back-to-back dimers interact to generate a tetramer with two double-stranded DNA cleavage modules. Each cleavage module contains two active sites facing each other, enabling double-strand DNA cuts. Biochemical, mutagenesis and structural characterization suggest three different monomers of the tetramer may be involved respectively in binding the modified cytosine, making the first proximal N12 cleavage in the same strand and then the second distal N16 cleavage in the opposite strand. Both cleavage events require binding of at least a second recognition site either in cis or in trans.

Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

PvuRts1I is a modification-dependent restriction endonuclease that recognizes 5-hydroxymethylcytosine (5hmC) as well as 5-glucosylhydroxymethylcytosine (5ghmC) in double-stranded DNA. Using PvuRts1I as the founding member, we define a family of homologous proteins with similar DNA modification-dependent recognition properties. At the sequence level, these proteins share a few uniquely conserved features. We show that these enzymes introduce a double-stranded cleavage at the 3′-side away from the recognized modified cytosine. The distances between the cleavage sites and the modified cytosine are fixed within a narrow range, with the majority being 11–13 nt away in the top strand and 9–10 nt away in the bottom strand. The recognition sites of these enzymes generally require two cytosines on opposite strand around the cleavage sites, i.e. 5′-CN11–13↓N9–10G-3′/3′-GN9–10↓N11–13C-5′, with at least one cytosine being modified for efficient cleavage. As one potential application for these enzymes is to provide useful tools for selectively mapping 5hmC sites, we have compared the relative selectivity of a few PvuRts1I family members towards different forms of modified cytosines. Our results show that the inherently different relative selectivity towards modified cytosines can have practical implications for their application. By using AbaSDFI, a PvuRts1I homolog with the highest relative selectivity towards 5ghmC, to analyze rat brain DNA, we show it is feasible to map genomic 5hmC sites close to base resolution. Our study offers unique tools for determining more accurate hydroxymethylomes in mammalian cells.

The MspJI family of modification-dependent restriction endonucleases for epigenetic studies

MspJI is a novel modification-dependent restriction endonuclease that cleaves at a fixed distance away from the modification site. Here, we present the biochemical characterization of several MspJI homologs, including FspEI, LpnPI, AspBHI, RlaI, and SgrTI. All of the enzymes specifically recognize cytosine C5 modification (methylation or hydroxymethylation) in DNA and cleave at a constant distance (N(12)/N(16)) away from the modified cytosine. Each displays its own sequence context preference, favoring different nucleotides flanking the modified cytosine. By cleaving on both sides of fully modified CpG sites, they allow the extraction of 32-base long fragments around the modified sites from the genomic DNA. These enzymes provide powerful tools for direct interrogation of the epigenome. For example, we show that RlaI, an enzyme that prefers (m)CWG but not (m)CpG sites, generates digestion patterns that differ between plant and mammalian genomic DNA, highlighting the difference between their epigenomic patterns. In addition, we demonstrate that deep sequencing of the digested DNA fragments generated from these enzymes provides a feasible method to map the modified sites in the genome. Altogether, the MspJI family of enzymes represent appealing tools of choice for method development in DNA epigenetic studies.