Tailoring the activity of restriction endonuclease PleI by PNA-induced DNA looping

DNA looping is one of the key factors allowing proteins bound to different DNA sites to signal one another via direct contacts. We demonstrate that DNA looping can be generated in an arbitrary chosen site by sequence-directed targeting of double-stranded DNA with pseudocomplementary peptide-nucleic acids (pcPNAs). We designed pcPNAs to mask the DNA from cleavage by type IIs restriction enzyme PleI while not preventing the enzyme from binding to its primary DNA recognition site. Direct interaction between two protein molecules (one bound to the original recognition site and the other to a sequence-degenerated site) results in a totally new activity of PleI: it produces a nick near the degenerate site. The PNA-induced nicking efficiency varies with the distance between the two protein-binding sites in a phase with the DNA helical periodicity. Our findings imply a general approach for the fine-tuning of proteins bound to DNA sites well separated along the DNA chain.     

Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

A new generation of PNAs, so-called pseudocomplementary PNAs (pcPNAs), which are able to target the designated sites on duplex DNA with mixed sequence of purines and pyrimidines via double-duplex invasion mode, has recently been introduced. It has been demonstrated that appropriate pairs of decameric pcPNAs block an access of RNA polymerase to the corresponding promoter. Here, we show that this type of PNAs protects selected DNA sites containing all four nucleobases from the action of restriction enzymes and DNA methyltransferases. We have found that pcPNAs as short as octamers form stable and sequence-specific complexes with duplex DNA in a very salt-dependent manner. In accord with a strand-invasion mode of complex formation, the pcPNA binding proceeds much faster with supercoiled than with linear plasmids. The double-duplex invasion complexes selectively shield specific DNA sites from BclI restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate DNA duplexes in a virtually sequence-unrestricted manner.     

Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences by Watson–Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules.     

Natural and engineered nicking endonucleases—from cleavage mechanism to engineering of strand-specificity

Restriction endonucleases (REases) are highly specific DNA scissors that have facilitated the development of modern molecular biology. Intensive studies of double strand (ds) cleavage activity of Type IIP REases, which recognize 4–8 bp palindromic sequences, have revealed a variety of mechanisms of molecular recognition and catalysis. Less well-studied are REases which cleave only one of the strands of dsDNA, creating a nick instead of a ds break. Naturally occurring nicking endonucleases (NEases) range from frequent cutters such as Nt.CviPII (^CCD; ^ denotes the cleavage site) to rare-cutting homing endonucleases (HEases) such as I-HmuI. In addition to these bona fida NEases, individual subunits of some heterodimeric Type IIS REases have recently been shown to be natural NEases. The discovery and characterization of more REases that recognize asymmetric sequences, particularly Types IIS and IIA REases, has revealed recognition and cleavage mechanisms drastically different from the canonical Type IIP mechanisms, and has allowed researchers to engineer highly strand-specific NEases. Monomeric LAGLIDADG HEases use two separate catalytic sites for cleavage. Exploitation of this characteristic has also resulted in useful nicking HEases. This review aims at providing an overview of the cleavage mechanisms of Types IIS and IIA REases and LAGLIDADG HEases, the engineering of their nicking variants, and the applications of NEases and nicking HEases.     

Isolation and characterization of a HpyC1I restriction-modification system in Helicobacter pylori

Using transposon shuttle mutagenesis, we identified six Helicobacter pylori mutants from the NTUH-C1 strain that exhibited decreased adherence and cell elongation. Inverse polymerase chain reaction and DNA sequencing revealed that the same locus was interrupted in these six mutants. Nucleotide and amino acid sequences showed no homologies with H. pylori 26695 and J99 strains. This novel open reading frame contained 1617 base pairs. The amino acid sequence shared 24% identity with a putative nicking enzyme in Bacillus halodurans and 23 and 20% identity with type IIS restriction endonucleases PleI and MlyI, respectively. The purified protein, HpyC1I, showed endonuclease activity with the recognition and cleavage site 5'-CCATC(4/5)-3'. Two open reading frames were located upstream of the gene encoding HpyC1I. Together, HpyC1I and these two putative methyltransferases (M1.HpyC1I and M2.HpyC1I) function as a restriction-modification (R-M) system. The HpyC1I R-M genes were found in 9 of the 15 H. pylori strains tested. When compared with the full genome, significantly lower G + C content of HpyC1I R-M genes implied that these genes might have been acquired by horizontal gene transfer. Plasmid DNA transformation efficiencies and chromosomal DNA digestion assays demonstrated protection from HpyC1I digestion by the R-M system. In conclusion, we have identified a novel R-M system present in approximately 60% of H. pylori strains. Disruption of this R-M system results in cell elongation and susceptibility to HpyC1I digestion.     

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction–modification (R–M) systems are classified into four groups. Type III R–M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25–27 bp downstream of one of the recognition sites). Like the Type I R–M enzymes, Type III R–M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R–M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R–M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.     

Site-selective and hydrolytic two-strand scission of double-stranded DNA using Ce(IV)/EDTA and pseudo-complementary PNA

By combining Ce(IV)/EDTA with two pseudo-complementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were site-selectively hydrolyzed at the target site. Either plasmid DNA (4361 bp) or its linearized form was used as the substrate. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was free from Watson–Crick base pairing with the counterpart DNA or the pcPNA. Upon the treatment of this invasion complex with Ce(IV)/EDTA at 37°C and pH 7.0, both of these single-stranded portions were selectively hydrolyzed at the designated site, resulting in the site-selective two-strand scission of the double-stranded DNA. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using ligase. The potential of these artificial systems for manipulation of huge DNA has been indicated.     

Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA

A monophosphate group was attached to the terminus of pseudo-complementary peptide nucleic acid (pcPNA), and two of thus modified pcPNAs were combined with Ce(IV)/EDTA for site-selective hydrolysis of double-stranded DNA. The site-selective DNA scission was notably accelerated by this chemical modification of pcPNAs. These second-generation artificial restriction DNA cutters (ARCUTs) differentiated the target sequence so strictly that no scission occurred even when only one DNA base-pair was altered to another. By using two of the activated ARCUTs simultaneously, DNA substrate was selectively cut at two predetermined sites, and the desired fragment was clipped and cloned. The DNA scission by ARCUT was also successful even when the target site was methylated by methyltransferase and protected from the corresponding restriction enzyme. Furthermore, potentiality of ARCUT for manipulation of huge DNA has been substantiated by site-selective scission of genomic DNA of Escherichia coli (composed of 4,600,000bp) at the target site. All these results indicate promising applications of ARCUTs for versatile purposes.     

Unidirectional translocation from recognition site and a necessary interaction with DNA end for cleavage by Type III restriction enzyme

Type III restriction enzymes have been demonstrated to require two unmethylated asymmetric recognition sites oriented head-to-head to elicit double-strand break 25–27 bp downstream of one of the two sites. The proposed DNA cleavage mechanism involves ATP-dependent DNA translocation. The sequence context of the recognition site was suggested to influence the site of DNA cleavage by the enzyme. In this investigation, we demonstrate that the cleavage site of the R.EcoP15I restriction enzyme does not depend on the sequence context of the recognition site. Strikingly, this study demonstrates that the enzyme can cleave linear DNA having either recognition sites in the same orientation or a single recognition site. Cleavage occurs predominantly at a site proximal to the DNA end in the case of multiple site substrates. Such cleavage can be abolished by the binding of Lac repressor downstream (3′ side) but not upstream (5′ side) of the recognition site. Binding of HU protein has also been observed to interfere with R.EcoP15I cleavage activity. In accordance with a mechanism requiring two enzyme molecules cooperating to elicit double-strand break on DNA, our results convincingly demonstrate that the enzyme translocates on DNA in a 5′ to 3′ direction from its recognition site and indicate a switch in the direction of enzyme motion at the DNA ends. This study demonstrates a new facet in the mode of action of these restriction enzymes.     

Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease

Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.     

Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I

DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.     

Characterization of EndoTT,a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis

EndoTT encoded by tte0829 of Thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA in vitro as well as binding dsDNA. In the presence of a low concentration of NaCl, EndoTT cleaved ss regions of damaged dsDNA efficiently but did not cleave DNA that was entirely ss or ds. At high concentrations of NaCl or MgCl₂ or ATP, there was also specific cleavage of ssDNA. This suggested a preference for ss/ds junctions to stimulate cleavage of the DNA substrates. EndoTT has six specific sites (a–f) in the oriC region (1–70 nt) of T. tengcongensis. Substitutions of nucleotides around site c prevented cleavage by EndoTT of both sites c and d, implying that the cleavage specificity may depend on both the nucleotide sequence and the secondary structure of the ssDNA. A C-terminal sub-fragment of EndoTT (residues 107–216) had both endonucleolytic and DNA-binding activity, whereas an N-terminal sub-fragment (residues 1–110) displayed only ssDNA-binding activity. Site-directed mutations showed that G¹⁷⁰, R¹⁷² and G¹⁷⁷ are required for the endonuclease activity of EndoTT, but not for DNA-binding, whereas D¹⁷¹, R¹⁷⁸ and G¹⁸⁹ are partially required for the DNA-binding activity.     

Hot-spot consensus of fluoroquinolone-mediated DNA cleavage by Gram-negative and Gram-positive type II DNA topoisomerases

Bacterial DNA gyrase and topoisomerase IV are selective targets of fluoroquinolones. Topoisomerase IV versus gyrase and Gram-positive versus Gram-negative behavior was studied based on the different recognition of DNA sequences by topoisomerase–quinolone complexes. A careful statistical analysis of preferred bases was performed on a large number (>400) of cleavage sites. We found discrete preferred sequences that were similar when using different enzymes (i.e. gyrase and topoisomerase IV) from the same bacterial source, but in part diverse when employing enzymes from different origins (i.e. Escherichia coli and Streptococcus pneumoniae). Subsequent analysis on the wild-type and mutated consensus sequences showed that: (i) Gn/Cn-rich sequences at and around the cleavage site are hot spots for quinolone-mediated strand breaks, especially for E. coli topoisomerases: we elucidated positions required for quinolone and enzyme recognition; (ii) for S. pneumoniae enzymes only, A and T at positions −2 and +6 are discriminating cleavage determinants; (iii) symmetry of the target sequence is a key trait to promote cleavage and (iv) the consensus sequence adopts a heteronomous A/B conformation, which may trigger DNA processing by the enzyme–drug complex.     

Type III restriction endonucleases translocate DNA in a reaction driven by recognition site-specific ATP hydrolysis.

Type III restriction/modification systems recognize short non-palindromic sequences, only one strand of which can be methylated. Replication of type III-modified DNA produces completely unmethylated recognition sites which, according to classical mechanisms of restriction, should be signals for restriction. We have shown previously that suicidal restriction by the type III enzyme EcoP15I is prevented if all the unmodified sites are in the same orientation: restriction by EcoP15I requires a pair of unmethylated, inversely oriented recognition sites. We have now addressed the molecular mechanism of site orientation-specific DNA restriction. EcoP15I is demonstrated to possess an intrinsic ATPase activity, the potential driving force of DNA translocation. The ATPase activity is uniquely recognition site-specific, but EcoP15I-modified sites also support the reaction. EcoP15I DNA restriction patterns are shown to be predetermined by the enzyme-to-site ratio, in that site-saturating enzyme levels elicit cleavage exclusively between the closest pair of head-to-head oriented sites. DNA restriction is blocked by Lac repressor bound in the intervening sequence between the two EcoP15I sites. These results rule out DNA looping and strongly suggest that cleavage is triggered by the close proximity of two convergently tracking EcoP15I-DNA complexes.     

Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification

A simple isothermal nucleic-acid amplification reaction, primer generation–rolling circle amplification (PG–RCA), was developed to detect specific nucleic-acid sequences of sample DNA. This amplification method is achievable at a constant temperature (e.g. 60°C) simply by mixing circular single-stranded DNA probe, DNA polymerase and nicking enzyme. Unlike conventional nucleic-acid amplification reactions such as polymerase chain reaction (PCR), this reaction does not require exogenous primers, which often cause primer dimerization or non-specific amplification. Instead, ‘primers’ are generated and accumulated during the reaction. The circular probe carries only two sequences: (i) a hybridization sequence to the sample DNA and (ii) a recognition sequence of the nicking enzyme. In PG–RCA, the circular probe first hybridizes with the sample DNA, and then a cascade reaction of linear rolling circle amplification and nicking reactions takes place. In contrast with conventional linear rolling circle amplification, the signal amplification is in an exponential mode since many copies of ‘primers’ are successively produced by multiple nicking reactions. Under the optimized condition, we obtained a remarkable sensitivity of 84.5 ymol (50.7 molecules) of synthetic sample DNA and 0.163 pg (~60 molecules) of genomic DNA from Listeria monocytogenes, indicating strong applicability of PG–RCA to various molecular diagnostic assays.     

Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

BspQI is a thermostable Type IIS restriction endonuclease (REase) with the recognition sequence 5′GCTCTTC N1/N4 3′. Here we report the cloning and expression of the bspQIR gene for the BspQI restriction enzyme in Escherichia coli. Alanine scanning of the BspQI charged residues identified a number of DNA nicking variants. After sampling combinations of different amino acid substitutions, an Nt.BspQI triple mutant (E172A/E248A/E255K) was constructed with predominantly top-strand DNA nicking activity. Furthermore, a triple mutant of BspQI (Nb.BspQI, N235A/K331A/R428A) was engineered to create a bottom-strand nicking enzyme. In addition, we demonstrated the application of Nt.BspQI in optical mapping of single DNA molecules. Nt or Nb.BspQI-nicked dsDNA can be further digested by E. coli exonuclease III to create ssDNA for downstream applications. BspQI contains two potential catalytic sites: a top-strand catalytic site (Ct) with a D-H-N-K motif found in the HNH endonuclease family and a bottom-strand catalytic site (Cb) with three scattered Glu residues. BlastP analysis of proteins in GenBank indicated a putative restriction enzyme with significant amino acid sequence identity to BspQI from the sequenced bacterial genome Croceibacter atlanticus HTCC2559. This restriction gene was amplified by PCR and cloned into a T7 expression vector. Restriction mapping and run-off DNA sequencing of digested products from the partially purified enzyme indicated that it is an EarI isoschizomer with 6-bp recognition, which we named CatHI (CTCTTC N1/N4).     

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA.

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.     

Functional cooperation between exonucleases and endonucleases--basis for the evolution of restriction enzymes

Many types of restriction enzymes cleave DNA away from their recognition site. Using the type III restriction enzyme, EcoP15I, which cleaves DNA 25–27 bp away from its recognition site, we provide evidence to show that an intact recognition site on the cleaved DNA sequesters the restriction enzyme and decreases the effective concentration of the enzyme. EcoP15I restriction enzyme is shown here to perform only a single round of DNA cleavage. Significantly, we show that an exonuclease activity is essential for EcoP15I restriction enzyme to perform multiple rounds of DNA cleavage. This observation may hold true for all restriction enzymes cleaving DNA sufficiently far away from their recognition site. Our results highlight the importance of functional cooperation in the modulation of enzyme activity. Based on results presented here and other data on well-characterised restriction enzymes, a functional evolutionary hierarchy of restriction enzymes is discussed.