Specific Endonuclease Research Articles

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA.

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5´-OH ends or 5´-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5´-end processing, DNA damage, and DNA repair.

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5´-OH ends or 5´-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5´-end processing, DNA damage, and DNA repair.

Abstract PR01: A functional variomic chemi-genetic screen in C. elegans identifies new synthetic lethal interactions with PARP inhibition that are conserved from worm to human

Abstract Poly ADP-ribose polymerase (PARP) inhibitors are anti-cancer therapeutics that are synthetic lethal (SL) with mutations affecting the homologous recombination genes BRCA1 or BRCA2. Previous work from our lab found that cohesin mutations, which are common in a range of tumour types, also confer sensitivity to PARP inhibition. We want to extend the SL profile of PARP inhibitors to identify the full spectrum of genetic variants that result in sensitivity to PARP inhibitors alone or in combination with DNA damaging agents. To this end, we are using the model organism Caenorhabditis elegans to screen for new SL interactions with PARP inhibitors. C. elegans provides an excellent in vivo system with which to investigate SL interactions with cancer-associated mutations, as many of the pathways and genes implicated in cancer are conserved between humans and C. elegans. In many ways, C. elegans is an animal model that combines the technical advantages of a single celled organism, such as yeast, with increased developmental complexity and a gene complement more akin to humans. We have developed a high throughput in vivo variomics approach using the C. elegans Million Mutation Project strain collection to screen 2,000 mutagenized and sequenced strains containing >800,000 homozygous genetic variations. To date we have found 16 strains that were sensitive to the PARP inhibitor olaparib. Frequency analysis of the variants in the sensitive strains identified multiple mutations affecting mus-81 and eme-1, which comprise the structure specific Mus81 endonuclease. Further investigation of the interaction with the Mus81 endonuclease mutants found that PARP inhibitor-treated mus-81 and eme-1 mutants exhibit profound somatic proliferation defects characterized by persistent anaphase bridges and that the mus-81 PARP inhibitor-mediated synthetic lethality was not suppressed by loss of non-homologous end joining proteins. We found that the synthetic lethality between MUS81 mutations and PARP inhibitor treatment is conserved in human cells. Similar to C. elegans, PARP inhibitor treated MUS81 knockout cells accumulate anaphase bridges and exhibit a phenotype distinct from PARP inhibitor treated BRCA2 knockout cells. Our variomic screening approach has identified new SL interactions with PARP inhibitors and has expanded our understanding of the mechanism underlying PARP inhibitor-mediated synthetic lethality. Citation Format: Nigel J. O’Neil, Melanie L. Bailey, Philip Hieter. A functional variomic chemi-genetic screen in C. elegans identifies new synthetic lethal interactions with PARP inhibition that are conserved from worm to human [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr PR01.

Association of functional SNP-1562C > T in MMP9 promoter with proliferative diabetic retinopathy in north Indian type 2 diabetes mellitus patients

ObjectiveRetinal angiogenesis is a hallmark of diabetic retinopathy. Matrix Metalloproteinases (MMPs) are involved in degradation of extracellular matrix (ECM). Functional SNP-1562C>T in the promoter of the MMP-9 gene results increase in transcriptional activity. The present work was designed to evaluate the contribution of functional SNP-1562C>T of MMP-9 gene to the risk of proliferative diabetic retinopathy (PDR) in type 2 diabetes mellitus (T2DM) patients in north Indian Population. MethodsThis Case control study comprised of a total of 645 individuals in which 320 were T2DM patients out of which 73 had PDR, 98 had non- proliferative diabetic retinopathy (NPDR), 149 T2DM cases without any eye related disease (DM) and 325 non diabetic healthy individuals as controls (non DM controls). Genotyping for SNP-1562C>T of MMP-9 was done by polymerase chain reactions followed by restriction analyses with specific endonucleases (PCR-RFLP). DNA sequencing was used to ascertain PCR-RFLP results. ResultsT allele frequency in PDR patients was 32.1%, 20.4% in NPDR, 15.4% in DM and 13.7% in controls. Statistically significant difference was observed in both allele and genotype distribution between the PDR versus non-DM control group (p<0.0001 by T allele; p=0.002 by TT and p<0.0001 by CT genotype). ConclusionsThe present study suggests that the functional SNP-1562C>T in the promoter of the MMP-9 gene could be regarded as a major risk factor for PDR as increased MMP-9 production from high expressing T allele may promote retinal angiogenesis.

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

BackgroundCytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material.MethodsWe established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species.ResultsAs a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice.ConclusionsPlant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations.

Structural dynamics of wild type and mutated forms of human L1 endonuclease and insights into its sequence specific nucleic acid binding mechanism: A molecular dynamics study

Biomolecular recognition of proteins and nucleic acids is mainly mediated by their structural features and the molecular dynamics simulations approach has been used to explore this recognition processes at the atomic level. L1-Endonuclease, an enzyme involved in L1 retrotransposition, cleaves the TA junction DNA (5′-TTTT/AA-3′) and expresses high specificity for target site recognition. The present study highlights the structural features of L1-endonuclease as well as DNA responsible for such specific recognition. Especially, the importance of βB6-B5 hairpin loop in DNA recognition has been elucidated by analyzing the dynamics of Thr192 mutated L1-endonuclease. In addition, simulations of the endonuclease complexed with DNA substrates (sequences having TA and CG junctions) revealed the specificity of L1 endonuclease towards TA junction. Molecular dynamics simulations revealed that the βB6-B5 hairpin loop protrudes well into the minor groove of DNA having TA junction and induces DNA bending such that the width of minor groove is increased. Such endonuclease induced bending of TA junction DNA sequence positions the scissile phosphodiester bond of DNA for cleavage. The innate property of minor groove widening in TA junction than in CG junction is utilized by the βB6-βB5 hairpin loop of endonuclease while recognizing the DNA sequences. The present study also highlights the role of Mg2+ cation in catalysis and attempts to explore the possible target site DNA cleavage mechanism.

Abstract 5367: Ngago: no evidence of targeted endonuclease activity

Abstract The development of a versatile and easy to use genome editing tool based on the bacterial CRISPR/Cas9 system has led to a vast increase in applications of genome editing in laboratories around the world. As an extremely powerful and versatile tool it has facilitated important discoveries in both basic and translational research as well as in a potentially wide range of therapeutic applications. Natronobacterium gregoryi Argonaute (NgAgo) protein was described as a favorable alternative to Cas9 due to its simplicity and higher endonuclease specificity. Thus far, there are no formal reports documenting the success of NgAgo in other laboratory settings and cellular systems. Utilizing the identical cellular background and protocol that was made publicly available by the authors of the initial study, we were unable to reproduce the endonuclease function of NgAgo. These results do not support the role of NgAgo in gene editing experiments. Note: This abstract was not presented at the meeting. Citation Format: Sabine Topka, Sara Kazzaz, Kenneth Offit, Vijai Joseph. Ngago: no evidence of targeted endonuclease activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5367. doi:10.1158/1538-7445.AM2017-5367

Lysine methylation of FEN1 by SET7 is essential for its cellular response to replicative stress.

The DNA damage response (DDR) is central to the cell survival and it requires post-translational modifications, in part, to sense the damage, amplify the signaling response and recruit and regulate DNA repair enzymes. Lysine methylation of histones such as H4K20 and non-histone proteins including p53 has been shown to be essential for the mounting of the DDR. It is well-known that the lysine methyltransferase SET7 regulates the DDR, as cells lacking this enzyme are hypersensitive to chemotherapeutic drugs. To define addition substrates of SET7 involved in the DDR, we screened a peptide array encompassing potential lysine methylation sites from >100 key DDR proteins and identified peptides from 58 proteins to be lysine methylated defining a methylation consensus sequence of [S>K-2; S>R-1; K0] consistent with previous findings. We focused on K377 methylation of the Flap endonuclease 1 (FEN1), a structure specific endonuclease with important functions in Okazaki fragment processing during DNA replication as a substrate of SET7. FEN1 was monomethylated by SET7 in vivo in a cell cycle dependent manner with levels increasing as cells progressed through S phase and decreasing as they exited S phase, as detected using K377me1 specific antibodies. Although K377me1 did not affect the enzymatic activity of FEN1, it was required for the cellular response to replicative stress by FEN1. These finding define FEN1 as a new substrate of SET7 required for the DDR.

In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases.

RNA editing is an essential post-transcriptional process that creates functional mitochondrial mRNAs in Kinetoplastids. Multiprotein editosomes catalyze pre-mRNA cleavage, uridine (U) insertion or deletion, and ligation as specified by guide RNAs. Three functionally and compositionally distinct editosomes differ by the mutually exclusive presence of the KREN1, KREN2 or KREN3 endonuclease and their associated partner proteins. Because endonuclease cleavage is a likely point of regulation for RNA editing, we elucidated endonuclease specificity in vivo. We used a mutant gamma ATP synthase allele (MGA) to circumvent the normal essentiality of the editing endonucleases, and created cell lines in which both alleles of one, two or all three of the endonucleases were deleted. Cells lacking multiple endonucleases had altered editosome sedimentation on glycerol gradients and substantial defects in overall editing. Deep sequencing analysis of RNAs from such cells revealed clear discrimination by editosomes between sites of deletion versus insertion editing and preferential but overlapping specificity for sites of insertion editing. Thus, endonuclease specificities in vivo are distinct but with some functional overlap. The overlapping specificities likely accommodate the more numerous sites of insertion versus deletion editing as editosomes collaborate to accurately edit thousands of distinct editing sites in vivo.

Loss of Caspase-Activated DNase Protects Against Atherosclerosis in Apolipoprotein E-Deficient Mice.

BackgroundAtherosclerosis is a chronic disease that is closely related to inflammation and macrophage apoptosis, which leads to secondary necrosis and proinflammatory responses in advanced lesions. Caspase‐activated DNase (CAD) is a double‐strand specific endonuclease that leads to the subsequent degradation of chromosome DNA during apoptosis. However, whether CAD is involved in the progression of atherosclerosis remains elusive.Methods and Results CAD −/−ApoE−/− and ApoE−/− littermates were fed a high‐fat diet for 28 weeks to develop atherosclerosis. Human specimens were collected from coronary heart disease (CHD) patients who were not suitable for transplantation. CAD expression was increased in the atheromatous lesions of CHD patients and high‐fat diet‐treated ApoE‐deficient mice. Further investigation demonstrated that CAD deficiency inhibited high‐fat diet‐induced atherosclerosis, as evidenced by decreased atherosclerotic plaques, inhibited inflammatory response, and macrophage apoptosis, as well as enhanced stability of plaques in CAD −/−ApoE−/− mice compared to the ApoE−/− controls. Bone marrow transplantation verified the effect of CAD on atherosclerosis from macrophages. Mechanically, the decrease in the phosphorylated levels of mitogen‐activated protein kinase (MAPK) kinase/extracellular signal‐regulated kinase 1 and 2 (MEK‐ERK1/2) that resulted from CAD knockout and the activation of nuclear factor kappa B signaling mediated by CAD stimulation that was suppressed by inhibiting ERK1/2 phosphorylation revealed the potential association between the role of CAD in atherosclerosis and the MAPK signaling pathway.ConclusionsIn conclusion, CAD deficiency protects against atherosclerosis through inhibiting inflammation and macrophage apoptosis, which is partially through inactivation of the MEK‐ERK1/2 signaling pathway. This finding provides a promising therapeutic target for treating atherosclerosis.

Nucleolytic processing of aberrant replication intermediates by an Exo1-Dna2-Sae2 axis counteracts fork collapse-driven chromosome instability.

Problems during DNA replication underlie genomic instability and drive malignant transformation. The DNA damage checkpoint stabilizes stalled replication forks thus counteracting aberrant fork transitions, DNA breaks and chromosomal rearrangements. We analyzed fork processing in checkpoint deficient cells by coupling psoralen crosslinking with replication intermediate two-dimensional gel analysis. This revealed a novel role for Exo1 nuclease in resecting reversed replication fork structures and counteracting the accumulation of aberrant intermediates resembling fork cleavage products. Genetic analyses demonstrated a functional interplay of Exo1 with Mus81, Dna2 and Sae2 nucleases in promoting cell survival following replication stress, suggestive of concerted nucleolytic processing of stalled forks. While Mus81 and other Structure Specific Endonucleases do not contribute to obvious collapsed fork transitions, Dna2 promotes reversed fork resection likely by facilitating Exo1 access to nascent strands. Instead, Sae2 cooperates with Exo1 in counteracting putative fork cleavage events linked to double strand breaks formation and increased gross chromosomal rearrangement rates. Our data indicate that in checkpoint deficient cells diverse nuclease activities interface to eliminate aberrant replication intermediates and prevent chromosome instability.

Activation of the DNA Damage Response Is a Conserved Function of HIV-1 and HIV-2 Vpr That Is Independent of SLX4 Recruitment.

ABSTRACTThere has been extraordinary progress in understanding the roles of lentiviral accessory proteins in antagonizing host antiviral defense proteins. However, the precise primary function of the accessory gene Vpr remains elusive. Here we suggest that engagement with the DNA damage response is an important function of primate lentiviral Vpr proteins because of its conserved function among diverse lentiviral lineages. In contrast, we show that, for HIV-1, HIV-2, and related Vpr isolates and orthologs, there is a lack of correlation between DNA damage response activation and interaction with the host SLX4 protein complex of structure specific endonucleases; some Vpr proteins are able to interact with SLX4, but the majority are not. Using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 method to knock out SLX4, we formally showed that HIV-1 and HIV-2 Vpr orthologs can still activate the DNA damage response and cell cycle arrest in the absence of SLX4. Together, our data suggest that activation of the DNA damage response, but not SLX4 interaction, is conserved and therefore indicative of an important function of Vpr. Our data also indicate that Vpr activates the DNA damage response through an SLX4-independent mechanism that remains uncharacterized.

In vivo genome editing as a potential treatment strategy for inherited retinal dystrophies

Invivo genome editing represents an emerging field in the treatment of monogenic disorders, as it may constitute a solution to the current hurdles in classic gene addition therapy, which are the low levels and limited duration of transgene expression. Following the introduction of a double strand break (DSB) at the mutational site by highly specific endonucleases, such as TALENs (transcription activator like effector nucleases) or RNA based nucleases (clustered regulatory interspaced short palindromic repeats - CRISPR-Cas), the cell's own DNA repair machinery restores integrity to the DNA strand and corrects the mutant sequence, thus allowing the cell to produce protein levels as needed. The DNA repair happens either through the error prone non-homologous end-joining (NHEJ) pathway or with high fidelity through homology directed repair (HDR) in the presence of a DNA donor template. A third pathway called microhomology mediated endjoining (MMEJ) has been recently discovered. In this review, the authors focus on the different DNA repair mechanisms, the current state of the art tools for genome editing and the particularities of the retina and photoreceptors with regard to invivo therapeutic approaches. Finally, current attempts in the field of retinal invivo genome editing are discussed and future directions of research identified.

An efficient and rapid method to detect and verify natural antisense transcripts of animal genes

High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lncRNA (long non-coding RNA), can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix (Xist antisense RNA), chicken LXN (latexin) and GFM1 (G elongation factor, mitochondrial 1), were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.

An ultrasensitive fluorescence method suitable for quantitative analysis of mung bean nuclease and inhibitor screening in vitro and vivo

Mung bean nuclease is a single stranded specific DNA and RNA endonuclease purified from mung bean sprouts. It yields 5′-phosphate terminated mono- and oligonucleotides. The activity level of this nuclease can act as a marker to monitor the developmental process of mung bean sprouts. In order to facilitate the activity and physiological analysis of this nuclease, we have developed a biosensing assay system based on the mung bean nuclease-induced single-stranded DNA scission and the affinity difference of graphene oxide for single-stranded DNA containing different numbers of bases. This end-point measurement method can detect mung bean nuclease in a range of 2×10−4 to 4×10−2 with a detection limit of 1×10−4 unit/mL. In addition, we demonstrate the utility of the assay for screening chemical antibiotics and metal ions, resulting in the identification of several inhibitors of this enzyme in vitro. Furthermore, we firstly report that inhibiting mung bean nuclease by gentamycin sulfate and kanamycin in vivo can suppress mung bean sprouts growth. In summary, this method provides an alternative tool for the biochemical analysis for mung bean nuclease and indicates the feasibility of high-throughput screening specific inhibitors of this nuclease in vitro and in vivo.

The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA.

CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described.

Unlocked Nucleic Acids for miRNA detection using two dimensional nano-graphene oxide

In this study we have used Unlocked Nucleic Acids (UNAs) to discriminate a breast cancer oncomiR from two other miRNAs in the same RNA family using two-dimensional graphene oxide nanoassemblies. Fluorescently labeled single stranded probe strands and graphene oxide nanoassemblies have been used to detect miR-10b and discriminate it from miR-10a, which differs by only a single nucleotide (12th base from the 5′ end), and miR-10c, which differs by only two nucleotides (12th and 16th bases from the 5′ end). We have determined the discrimination efficacy and detection capacity of a DNA probe with two inserted UNA monomers (UNA2), and compared it to the DNA probe with two purposefully inserted mutations (DNAM2) and full complementary sequence (DNAfull). We have observed that UNA2 is 50 times more powerful than DNAfull in discriminating miR-10b from miR-10c while generating an equally high fluorescence signal. This fluorescence signal was then further enhanced with the use of the highly specific endonuclease dsDNase for an enzymatic amplification step. The results demonstrate that the underutilized UNAs have enormous potential for miRNA detection and offer remarkable discrimination efficacy over single and double mismatches.

Identification of a conserved 5'-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair.

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5′-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2′-deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway. We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs.

An efficient gene targeting system using homologous recombination in plants

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.

Genome-wide mapping of embedded ribonucleotides and other noncanonical nucleotides using emRiboSeq and EndoSeq.

Ribonucleotides are the most common noncanonical nucleotides incorporated into the genome of replicating cells. They are efficiently removed by ribonucleotide excision repair initiated by RNase H2 cleavage. In the absence of RNase H2, such embedded ribonucleotides can be used to track DNA polymerase activity in vivo. To determine their precise location in Saccharomyces cerevisiae, we developed embedded ribonucleotide sequencing (emRiboSeq), which uses recombinant RNase H2 to selectively create ligatable 3'-hydroxyl groups, in contrast to alternative methods that use alkaline hydrolysis. EmRiboSeq allows reproducible, strand-specific and potentially quantitative detection of embedded ribonucleotides at single-nucleotide resolution. For the genome-wide mapping of other noncanonical bases, RNase H2 can be replaced with specific nicking endonucleases in this protocol; we term this method endonuclease sequencing (EndoSeq). With the protocol taking <5 d to complete, these methods allow the in vivo study of DNA replication and repair, including the identification of replication origins and termination regions.