Site-specific Cleavage Research Articles

Highly sensitive methyltransferase activity assay and inhibitor screening based on fluorescence quenching of graphene oxide integrated with the site-specific cleavage of restriction endonuclease.

We report a sensitive and selective approach for the DNA methyltransferase (MTase) activity assay and MTase inhibitor screening by coupling the fluorescence quenching of graphene oxide with site-specific cleavage of a restriction endonuclease.

ヘム鉄代謝の鍵となる電子伝達複合体の構造解析

Heme is an important co-factor for several biological processes such as respiration, electron transfer and enzymatic reaction, whereas free heme is toxic because of the production of reactive oxygen species. Therefore free heme should be decomposed immediately in vivo. Heme oxygenase (HO) is an indispensable player for heme degradation. HO catalyzes the site-specific cleavage of heme porphyrin ring to produce biliverdin, ferrous iron, and carbon monoxide. This reaction requires several oxygen molecules and electrons supplied from NADPH-cytochrome P450 reductase (CPR). We have determined the crystal structure of the redox complex of CPR and HO. In this structure, FMN bound to CPR is close to heme bound to HO, whereas FAD bound to CPR is far from both of FMN and heme. Electron transfer mechanism from CPR to HO with dynamic conformation change of CPR is discussed.

The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

A facile mechanophore functionalization of an amphiphilic block copolymer towards remote ultrasound and redox dual stimulus responsiveness

Two different site-specific cleavage mechanisms for copolymer micelles under ultrasound and redox were confirmed. The finding that ultrasound induced mechanochemical cleavage of central ester bonds can open up a new way in the design of novel ultrasound responsive copolymer micelles for drug release.

Identification and Functional Role of Calpain Cleavage Site in Na+-Ca2+ Exchanger 1 (NCX1)

Altered Ca2+ homeostasis is a key determinant of cardiac remodeling and contractility during chronic heart disease. Aberrant activation of calpain, a ubiquitous Ca2+-dependent protease can contribute to loss of Ca2+ control in cardiomyocytes. Calpain cleaves NCX1, but the underlying significance of the direct cleavage of NCX1 by calpain remains to be determined.By bioinformatics and mutational analysis we identified M369 as a putative calpain cleavage site residing within the α-catenin-like domain (CLD) in NCX1. Importantly, the cleavage of NCX1 at M369 corresponded to a proteolytic fragment of 75 kDa in left ventricular biopsies from aorta stenosis (AS) patients and in the failing left ventricle of rats following aortic banding (AB). Moreover, calpain binding in the CLD and Ca2+ -binding domain (CBD1) of NCX1 protein were identified by overlay assays. In order to investigate the specific function of M369 cleavage and avoid auxiliary and indirect functions of calpain, we utilized the protease Tobacco Etch Virus (TEV) to investigate site-specific cleavage. The TEV protease recognition site was inserted at the location of M369 in NCX1. By employing the patch clamp technique on transfected HEK cells, we observed a significant reduction of NCX1 current following TEV cleavage. In conclusion, we have identified and investigated the functional role of the calpain cleavage site at M369 in the CLD of NCX1. Our findings show that cleavage of the NCX1 at M369 reduces the total current. This reduction of NCX1 activity could potentially function to compensate for altered Ca2+ homeostasis associated with NCX upregulation during heart failure.

RNAi Factors Are Present and Active in Human Cell Nuclei

SummaryRNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP and TRNC6A/GW182 are in the human nucleus and associate together in multi-protein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements.

Production of an aminoterminally truncated, stable type of bioactive mouse fibroblast growth factor 4 in Escherichia coli

In mice, fibroblast growth factor 4 (Fgf4) is a crucial gene for the generation of trophectoderm, progenitor cells of the placenta. Therefore, exogenous FGF4 promotes the isolation and maintenance of trophoblast stem cells from preimplantation embryos. We previously produced a 6× histidine (His)-tagged, mouse FGF4 (Pro(31)-Leu(202)) without a secretory signal peptide at the amino-terminus, referred to as HismFGF4, in Escherichia coli. Here, we found that HismFGF4 was unstable, such as in phosphate-buffered saline. In these conditions, site-specific cleavage between Ser(50) and Leu(51) was identified. In order to generate stable mouse FGF4 derivatives, a 6× His-tagged mouse FGF4 (Leu(51)-Leu(202)), termed HismFGF4L, was expressed in E. coli. HismFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HismFGF4L was relatively stable. HismFGF4L exerted significant mitogenic activities at concentrations as low as 0.01 nM (P < 0.01) in mouse embryonic fibroblast Balb/c 3T3 cells expressing FGF receptor 2. In the presence of PD173074, an FGF receptor inhibitor, the growth-promoting activity of HismFGF4L was abolished. Taken together, we suggest that aminoterminally truncated HismFGF4L is capable of promoting the proliferation of mouse-derived cells via an authentic FGF signaling pathway. We consider that HismFGF4L is useful as a derivative of mouse FGF4 protein for analyzing the effects of mouse FGF4 and for stimulating cell growth of mouse-derived cells, such as trophoblast stem cells. Our study provides a simple method for the production of a bioactive, stable mouse FGF4 derivative in E. coli.

Combinatorial Engineering of 1-Deoxy-D-Xylulose 5-Phosphate Pathway Using Cross-Lapping In Vitro Assembly (CLIVA) Method

The ability to assemble multiple fragments of DNA into a plasmid in a single step is invaluable to studies in metabolic engineering and synthetic biology. Using phosphorothioate chemistry for high efficiency and site specific cleavage of sequences, a novel ligase independent cloning method (cross-lapping in vitro assembly, CLIVA) was systematically and rationally optimized in E. coli. A series of 16 constructs combinatorially expressing genes encoding enzymes in the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway were assembled using multiple DNA modules. A plasmid (21.6 kb) containing 16 pathway genes, was successfully assembled from 7 modules with high efficiency (2.0 x 103 cfu/ µg input DNA) within 2 days. Overexpressions of these constructs revealed the unanticipated inhibitory effects of certain combinations of genes on the production of amorphadiene. Interestingly, the inhibitory effects were correlated to the increase in the accumulation of intracellular methylerythritol cyclodiphosphate (MEC), an intermediate metabolite in the DXP pathway. The overexpression of the iron sulfur cluster operon was found to modestly increase the production of amorphadiene. This study demonstrated the utility of CLIVA in the assembly of multiple fragments of DNA into a plasmid which enabled the rapid exploration of biological pathways.

Evolution of CRISPR RNA recognition and processing by Cas6 endonucleases

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5′-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.

Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA

N(6)-methyladenosine (m(6)A) is the most abundant modification in mammalian mRNA and long noncoding RNA (lncRNA). Recent discoveries of two m(6)A demethylases and cell-type and cell-state-dependent m(6)A patterns indicate that m(6)A modifications are highly dynamic and likely play important biological roles for RNA akin to DNA methylation or histone modification. Proposed functions for m(6)A modification include mRNA splicing, export, stability, and immune tolerance; but m(6)A studies have been hindered by the lack of methods for its identification at single nucleotide resolution. Here, we develop a method that accurately determines m(6)A status at any site in mRNA/lncRNA, termed site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET). The method determines the precise location of the m(6)A residue and its modification fraction, which are crucial parameters in probing the cellular dynamics of m(6)A modification. We applied the method to determine the m(6)A status at several sites in two human lncRNAs and three human mRNAs and found that m(6)A fraction varies between 6% and 80% among these sites. We also found that many m(6)A candidate sites in these RNAs are however not modified. The precise determination of m(6)A status in a long noncoding RNA also enables the identification of an m(6)A-containing RNA structural motif.

Identification of Site-Specific Degradation in Bacterially Expressed Human Fibroblast Growth Factor 4 and Generation of an Aminoterminally Truncated, Stable Form

Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for tumorigenesis in humans and the development of mammalian embryos. The secreted, mature form of human FGF4 is thought to be comprised of 175 amino acid residues (proline(32) to leucine(206), Pro(32)-Leu(206)). Here, we found that bacterially expressed, 6× histidine (His)-tagged human FGF4 (Pro(32)-Leu(206)) protein, referred to as HishFGF4, was unstable such as in phosphate-buffered saline. In these conditions, site-specific cleavage, including between Ser(54) and Leu(55), in HishFGF4 was identified. In order to generate stable human FGF4 derivatives, a 6× His-tagged human FGF4 (Leu(55)-Leu(206)), termed HishFGF4L, was expressed in Escherichia coli. HishFGF4L could be purified from the supernatant of cell lysates by heparin column chromatography. In phosphate-buffered saline, HishFGF4L was considered as sufficiently stable. HishFGF4L exerted significant mitogenic activities in mouse embryonic fibroblast Balb/c 3T3 cells. In the presence of PD173074, an FGF receptor inhibitor, the growth-stimulating activity of HishFGF4L disappeared. Taken together, we suggest that HishFGF4L is capable of promoting cell growth via an authentic FGF signaling pathway. Our study provides a simple method for the production of a bioactive human FGF4 derivative in E. coli.

The Design and In Vivo Evaluation of Engineered I-OnuI-Based Enzymes for HEG Gene Drive

The homing endonuclease gene (HEG) drive system, a promising genetic approach for controlling arthropod populations, utilises engineered nucleases to spread deleterious mutations that inactivate individual genes throughout a target population. Previous work with a naturally occurring LAGLIDADG homing endonuclease (I-SceI) demonstrated its feasibility in both Drosophila and Anopheles. Here we report on the next stage of this strategy: the redesign of HEGs with customized specificity in order to drive HEG-induced ‘homing’ in vivo via break-induced homologous recombination. Variants targeting a sequence within the Anopheles AGAP004734 gene were created from the recently characterized I-OnuI endonuclease, and tested for cleavage activity and frequency of homing using a model Drosophila HEG drive system. We observed cleavage and homing at an integrated reporter for all endonuclease variants tested, demonstrating for the first time that engineered HEGs can cleave their target site in insect germline cells, promoting targeted mutagenesis and homing. However, in comparison to our previously reported work with I-SceI, the engineered I-OnuI variants mediated homing with a reduced frequency, suggesting that site-specific cleavage activity is insufficient by itself to ensure efficient homing. Taken together, our experiments take a further step towards the development of a viable HEG-based population control strategy for insects.

Graphene‐Mesoporous Silica‐Dispersed Palladium Nanoparticles‐Based Probe Carrier Platform for Electrocatalytic Sensing of Telomerase Activity at Less Than Single‐Cell Level

Human telomerase is a specialized ribonucleoprotein polymerase and is used as a clinical cancer marker and special target for chemotherapy. Traditional telomerase detection uses "telomerase repeat amplification protocol" (TRAP) assay. However, TRAP is questioned because it requires use of DNA polymerases, which is susceptible to polymerase chain reaction (PCR)-derived artifacts and time-consuming. Here, a novel PCR-free, electrocatalytic assay for signal-amplified detection of telomerase activity using graphene-mesoporous silica-dispersed palladium nanoparticles-based probe carrier platform to improve probe-target recognition is reported. The low background noise is achieved by using DNA site-specific cleavage endonuclease and the detection signal is amplified by using hemoglobin. As a highly efficient electron sink, hemoglobin does not carry out direct reduction at the surface, minimizing false positives. These merits make this assay highly sensitive, achieving the sensitivity comparable to TRAP. The developed electrocatalysis assay is PCR-free, simple in design, and fast in operation, therefore avoiding PCR amplification-related errors, which make it more reliable to evaluate telomerase activity for clinical use.

Site-specific Relaxase Activity of a VirD2-like Protein Encoded within the tfs4 Genomic Island of Helicobacter pylori

Four different type IV secretion systems are variously represented in the genomes of different Helicobacter pylori strains. Two of these, encoded by tfs3 and tfs4 gene clusters are contained within self-transmissible genomic islands. Although chromosomal excision of tfs4 circular intermediates is reported to be dependent upon the function of a tfs4-encoded XerD tyrosine-like recombinase, other factors required for transfer to a recipient cell have not been demonstrated. Here, we characterize the functional activity of a putative tfs4-encoded VirD2-like relaxase protein. Tfs4 VirD2 was purified as a fusion to maltose-binding protein and demonstrated to bind and nick both supercoiled duplex DNA and oligonucleotides in vitro in a manner dependent upon the presence of Mg(2+) but independently of any auxiliary proteins. Unusually, concentration-dependent nicking of duplex DNA appeared to require only transient protein-DNA interaction. Although phylogenetically distinct from established relaxase families, site-specific cleavage of oligonucleotides by Tfs4 VirD2 required the nick region sequence 5'-ATCCTG-3' common to transfer origins (oriT) recognized by MOBP conjugative relaxases. Cleavage resulted in covalent attachment of MBP-VirD2 to the 5'-cleaved end, consistent with conventional relaxase activity. Identification of an oriT-like sequence upstream of tfs4 virD2 and demonstration of VirD2 protein-protein interaction with a putative VirC1 relaxosome component indicate that transfer initiation of the tfs4 genomic island is analogous to mechanisms underlying mobilization of other integrated mobile elements, such as integrating conjugative elements, requiring site-specific targeting of relaxase activity to a cognate oriT sequence.

Structure of the NS1 Protein N-Terminal Origin Recognition/Nickase Domain from the Emerging Human Bocavirus

Human bocavirus is a newly identified, globally prevalent, parvovirus that is associated with respiratory infection in infants and young children. Parvoviruses encode a large nonstructural protein 1 (NS1) that is essential for replication of the viral single-stranded DNA genome and DNA packaging and may play versatile roles in virus-host interactions. Here, we report the structure of the human bocavirus NS1 N-terminal domain, the first for any autonomous parvovirus. The structure shows an overall fold that is canonical to the histidine-hydrophobic-histidine superfamily of nucleases, which integrates two distinct DNA-binding sites: (i) a positively charged region mediated by a surface hairpin (residues 190 to 198) that is responsible for recognition of the viral origin of replication of the double-stranded DNA nature and (ii) the nickase active site that binds to the single-stranded DNA substrate for site-specific cleavage. The structure reveals an acidic-residue-rich subdomain that is present in bocavirus NS1 proteins but not in the NS1 orthologs in erythrovirus or dependovirus, which may mediate bocavirus-specific interaction with DNA or potential host factors. These results provide insights into recognition of the origin of replication and nicking of DNA during bocavirus genome replication. Mapping of variable amino acid residues of NS1s from four human bocavirus species onto the structure shows a scattered pattern, but the origin recognition site and the nuclease active site are invariable, suggesting potential targets for antivirals against this clade of highly diverse human viruses.

Human Apurinic/Apyrimidinic Endonuclease 1

Human apurinic/apyrimidinic endonuclease 1 (APE1, also known as REF-1) was isolated based on its ability to cleave at AP sites in DNA or activate the DNA binding activity of certain transcription factors. We review herein topics related to this multi-functional DNA repair and stress-response protein. APE1 displays homology to Escherichia coli exonuclease III and is a member of the divalent metal-dependent α/β fold-containing phosphoesterase superfamily of enzymes. APE1 has acquired distinct active site and loop elements that dictate substrate selectivity, and a unique N-terminus which at minimum imparts nuclear targeting and interaction specificity. Additional activities ascribed to APE1 include 3'-5' exonuclease, 3'-repair diesterase, nucleotide incision repair, damaged or site-specific RNA cleavage, and multiple transcription regulatory roles. APE1 is essential for mouse embryogenesis and contributes to cell viability in a genetic background-dependent manner. Haploinsufficient APE1(+/-) mice exhibit reduced survival, increased cancer formation, and cellular/tissue hyper-sensitivity to oxidative stress, supporting the notion that impaired APE1 function associates with disease susceptibility. Although abnormal APE1 expression/localization has been seen in cancer and neuropathologies, and impaired-function variants have been described, a causal link between an APE1 defect and human disease remains elusive. Ongoing efforts aim at delineating the biological role(s) of the different APE1 activities, as well as the regulatory mechanisms for its intra-cellular distribution and participation in diverse molecular pathways. The determination of whether APE1 defects contribute to human disease, particularly pathologies that involve oxidative stress, and whether APE1 small-molecule regulators have clinical utility, is central to future investigations.

Zinc(II) complexes of Pro-Gly and Pro-Leu dipeptides: Synthesis, characterization, in vitro DNA binding and cleavage studies

Dipeptide (Pro-Gly and Pro-Leu) Zinc(II) complexes 1 and 2 were designed and synthesized for potential use as cancer chemotherapeutic agents. In order to augment the DNA recognition of metallonuclease activity, zinc metal ion was tethered to peptide motif to carry out DNA site specific hydrolytic cleavage. The structural formulation of the complexes 1 and 2 was done by elemental analysis, spectroscopic methods (IR, NMR, electronic) and molar conductance measurements. Their in vitro DNA binding profile was investigated by UV–vis titrations, fluorescence titrations and circular dichroism which revealed that these complexes bind to CT DNA by electrostatic interactions via groove binding mode. Zn(II) Pro-Gly complex 1 showed greater binding affinity to CT DNA as compared to the Zn(II) Pro-Leu complex 2 due to steric constraints in the latter. The supercoiled pBR322 DNA cleavage activity of complex 1, ascertained by gel electrophoresis demonstrated efficient DNA cleaving ability via hydrolytic mechanistic pathway. Further, the molecular docking studies confirmed that complex 1 bind to the minor groove of DNA having AT-rich sequences with relative binding energy of −196.72kJmol−1.

Use of a molybdenum(VI) complex as artificial protease in protein photocleavage

In this study, a molybdenum(VI) peroxo α-amino acid complex, MoO(O2)2(α-leucine) (H2O), was prepared and used as an artificial protease for site-specific cleavage of porcine pepsin, a model protein. Cleavage of pepsin by MoO(O2)2(α-leucine) (H2O) was achieved under photochemical conditions at room temperature and pH 7.0. The reaction was activated by irradiation of the MoO(O2)2(α-leucine) (H2O)-protein mixture by UV light (320 and 340nm) for up to 30min. No cleavage was observed in the absence of MoO(O2)2(α-leucine) (H2O) or the light. The photocleavage yield increased with irradiation time. The cleaved fragments were sequencable, and the cleavage site was assigned to Leu(112)–Tyr(113). The cleavage reaction was quenched by ethanol. Therefore, hydroxyl radicals may be involved in the reaction and responsible for the cleavage of the protein. This is the first demonstration of the successful photocleavage of proteins by a molybdenum complex. This observation can provide a new approach for the photochemical footprinting of metal binding sites on proteins.

Degradation of rRNA in BM-N cells from the silkworm Bombyx mori during abortive infection with heterologous nucleopolyhedroviruses

Cell lines derived from the silkworm, Bombyx mori, are only permissive for B. mori nucleopolyhedrovirus (NPV), with other NPVs generally resulting in abortive infection. Here, we demonstrate that rRNA of B. mori BM-N cells undergoes rapid degradation through site-specific cleavage upon infection with NPVs from Autographa californica (AcMNPV), Hyphantria cunea (HycuMNPV), Spodoptera exigua (SeMNPV) and Spodoptera litura (SpltMNPV). No significant decreases in cellular RNA were observed in Ld652Y, Se301, Sf9, SpIm and S2 cells infected with AcMNPV or HycuMNPV, indicating the response is unique to BM-N cells. A transient expression assay using a cosmid library of the HycuMNPV genome demonstrated that HycuMNPV P143 is responsible for rRNA degradation, which was also detected in BM-N cells transfected with plasmids expressing the P143 proteins from AcMNPV, SeMNPV and SpltMNPV. These results indicate that B. mori evolved to acquire a unique antiviral immune mechanism that is activated by P143 proteins from heterologous NPVs.