scholarly journals DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

2019 ◽  
Author(s):  
Jorrit W. Hegge ◽  
Daan C. Swarts ◽  
Stanley D. Chandradoss ◽  
Tao Ju Cui ◽  
Jeroen Kneppers ◽  
...  
Keyword(s):  
Host Defense ◽  
Dna Cleavage ◽  
Elevated Temperatures ◽  
Clostridium Butyricum ◽  
Dna Targeting ◽  
Dna Cleaving ◽  
Double Stranded Dna ◽  
Argonaute Proteins ◽  
Mesophilic Bacterium

AbstractProkaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5’-end and thymidines in the sub-seed segment (siDNA nucleotides 2-4). Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo–based applications.

scholarly journals Programmable Cleavage of Double-stranded DNA by Combined Action of Argonaute CbAgo from Clostridium butyricum and Nuclease Deficient RecBC Helicase from E.coli

2021 ◽  
Author(s):  
Rita Vaiskunaite ◽  
Jogirdas Vainauskas ◽  
Janna Morris ◽  
Vladimir Potapov ◽  
Jurate Bitinaite
Keyword(s):  
Dna Cleavage ◽  
Dna Helicase ◽  
Clostridium Butyricum ◽  
Dna Targeting ◽  
Double Stranded Dna ◽  
Dna Strands ◽  
Linear Dsdna ◽  
Combined Action ◽  
Dna Strand

Prokaryotic Argonautes (pAgos) use small nucleic acids as specificity guides to cleave single-stranded DNA at complementary sequences. DNA targeting function of pAgos creates attractive opportunities for DNA manipulations that require programmable DNA cleavage. Discovery of mesophilic Argonautes active at physiological temperature places pAgos closer to their possible application for genome editing as a simpler alternative to CRISPR/Cas nucleases. Currently, the use of mesophilic pAgos as programmable DNA endonucleases is hampered by their poor action on double-stranded DNA (dsDNA), mainly due to their inability to invade the DNA duplex. The present study demonstrates that efficient in vitro cleavage of double-stranded DNA by mesophilic Argonaute CbAgo from Clostridium butyricum can be activated via the DNA strand unwinding activity of nuclease deficient mutant of RecBC DNA helicase from Escherichia coli (referred to as RecBexo-C). Properties of CbAgo and characteristics of simultaneous cleavage of complementary DNA strands in concurrence with DNA strand unwinding by RecBexo-C were thoroughly explored using 0.3-25 kb DNA substrates. When combined with RecBexo-C helicase, CbAgo was capable of cleaving target sequences located 11-12.5 kb from the ends of linear dsDNA at 37 C. Our study demonstrates that CbAgo with RecBexo-C can be programmed to generate dsDNA fragments flanked with custom-designed single-stranded overhangs suitable for ligation with compatible DNA fragments. At present, the combination of CbAgo and RecBexo-C represents the most efficient mesophilic DNA-guided DNA-cleaving programmable endonuclease for use in diagnostic and synthetic biology methods that require sequence-specific nicking/cleavage of dsDNA at any desired location.

scholarly journals Cleavage of Supercoiled Circular Double-stranded DNA Induced by a Eukaryotic Cambialistic Superoxide Dismutase from Cinnamomum camphora

2004 ◽  
Vol 36 (9) ◽  
pp. 609-617 ◽  
Author(s):  
Bao-Zhong Wang ◽  
Xu-Bin Wei ◽  
Wang-Yi Liu
Keyword(s):  
Superoxide Dismutase ◽  
Hydroxyl Radical ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Tryptophan Residue ◽  
Cinnamomum Camphora ◽  
Supercoiled Dna ◽  
Linear Dna ◽  
Dna Cleaving ◽  
Double Stranded Dna

Abstract A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

scholarly journals Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

2019 ◽  
Author(s):  
Anton Kuzmenko ◽  
Denis Yudin ◽  
Sergei Ryazansky ◽  
Andrey Kulbachinskiy ◽  
Alexei A. Aravin
Keyword(s):  
Dna Cleavage ◽  
Elevated Temperatures ◽  
Biochemical Characterization ◽  
Clostridium Butyricum ◽  
Seed Region ◽  
Mesophilic Bacteria ◽  
Highly Active ◽  
Wide Range

ABSTRACTArgonaute (Ago) proteins are the key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleave of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In contrast to previously studied pAgos, CbAgo and LrAgo can use not only 5’-phosphorylated but also 5’-hydroxyl DNA guides, with diminished precision of target cleavage. Both LrAgo and CbAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3’-guide region. CbAgo is highly active under a wide range of conditions and can be used for programmable endonucleolytic cleavage of both single-stranded and double-stranded DNA substrates at moderate temperatures. The biochemical characterization of mesophilic pAgo proteins paths the way for their use for DNA manipulations both in vitro and in vivo.

scholarly journals Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

2021 ◽  
Vol 7 (11) ◽  
pp. eabd6030
Author(s):  
Isabel Strohkendl ◽  
Fatema A. Saifuddin ◽  
Bryan A. Gibson ◽  
Michael K. Rosen ◽  
Rick Russell ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Dna Cleavage ◽  
Genome Engineering ◽  
Eukaryotic Cells ◽  
Loop Formation ◽  
Chromatin Compaction ◽  
Dna Targeting ◽  
Genomic Regions ◽  
Dna Bendability ◽  
Nucleosome Arrays

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

scholarly journals A YoeB toxin cleaves both RNA and DNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Julia McGillick ◽  
Jessica R. Ames ◽  
Tamiko Murphy ◽  
Christina R. Bourne
Keyword(s):  
Dna Cleavage ◽  
Divalent Cations ◽  
Nuclease Activity ◽  
Dose Dependence ◽  
Cleavage Activity ◽  
Double Stranded Dna ◽  
Dna Cleavage Activity ◽  
Evolutionarily Conserved ◽  
Toxin Protein ◽  
Rna And Dna

AbstractType II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.

Optical Absorption Spectroscopy of DNA-Wrapped HiPco Carbon Nanotubes

2019 ◽  
Vol 943 ◽  
pp. 95-99
Author(s):  
Li Jun Wang ◽  
Kazuo Umemura
Keyword(s):  
Carbon Nanotubes ◽  
Aqueous Solution ◽  
Optical Absorption ◽  
Absorption Spectroscopy ◽  
Near Infrared ◽  
Nir Spectroscopy ◽  
Optical Characterization ◽  
Optical Absorption Spectroscopy ◽  
Double Stranded Dna

Optical absorption spectroscopy provides evidence for individually dispersed carbon nanotubes. A common method to disperse SWCNTs into aqueous solution is to sonicate the mixture in the presence of a double-stranded DNA (dsDNA). In this paper, optical characterization of dsDNA-wrapped HiPco carbon nanotubes (dsDNA-SWCNT) was carried out using near infrared (NIR) spectroscopy and photoluminescence (PL) experiments. The findings suggest that SWCNT dispersion is very good in the environment of DNA existing. Additionally, its dispersion depends on dsDNA concentration.

scholarly journals Characterization of a Novel Thermobifida fusca Bacteriophage P318

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 1042
Author(s):  
Cheepudom ◽  
Lin ◽  
Lee ◽  
Meng
Keyword(s):  
Plant Cell Wall ◽  
Hydrolytic Enzymes ◽  
Thermobifida Fusca ◽  
Genome Replication ◽  
Putative Orfs ◽  
Base Pairs ◽  
Double Stranded Dna ◽  
Virion Morphogenesis ◽  
Genome Information

Thermobifida fusca is of biotechnological interest due to its ability to produce an array of plant cell wall hydrolytic enzymes. Nonetheless, only one T. fusca bacteriophage with genome information has been reported to date. This study was aimed at discovering more relevant bacteriophages to expand the existing knowledge of phage diversity for this host species. With this end in view, a thermostable T. fusca bacteriophage P318, which belongs to the Siphoviridae family, was isolated and characterized. P318 has a double-stranded DNA genome of 48,045 base pairs with 3′-extended COS ends, on which 52 putative ORFs are organized into clusters responsible for the order of genome replication, virion morphogenesis, and the regulation of the lytic/lysogenic cycle. In comparison with T. fusca and the previously discovered bacteriophage P1312, P318 has a much lower G+C content in its genome except at the region encompassing ORF42, which produced a protein with unknown function. P1312 and P318 share very few similarities in their genomes except for the regions encompassing ORF42 of P318 and ORF51 of P1312 that are homologous. Thus, acquisition of ORF42 by lateral gene transfer might be an important step in the evolution of P318.

scholarly journals Analysis of Ribonucleotide 5′-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

2016 ◽  
Vol 61 (3) ◽  
Author(s):  
Gaofei Lu ◽  
Gregory R. Bluemling ◽  
Paul Collop ◽  
Michael Hager ◽  
Damien Kuiper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Zika Virus ◽  
Nonstructural Protein ◽  
Rna Dependent Rna Polymerase ◽  
Antiviral Compounds ◽  
Double Stranded Dna ◽  
Virus Rna ◽  
Potential Inhibitors

ABSTRACT Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primer-template pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn2+ is required for enzymatic activity, while Mg2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

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