scholarly journals EXTENDED TUDOR-DOMAINS of the piRNA BIOGENESIS PATHWAY HAVE RNA-SPECIFIC NUCLEASE ACTIVITY

2021 ◽  
Author(s):  
Neha Dhimole ◽  
Susanne Zur Lage ◽  
Wilfried Klug ◽  
Teresa Carlomagno
Keyword(s):  
Nuclease Activity ◽  
Protein Fold ◽  
Endonuclease Activity ◽  
Deleterious Mutations ◽  
The Core ◽  
Argonaute Proteins ◽  
Biochemical Assays ◽  
Tightly Coupled ◽  
Amplification Loop ◽  
Prime End

piRNAs are essential for transposon repression and protecting the germline from deleterious mutations. piRNA biogenesis comprises a primary and secondary pathway, and involves PIWI clade argonaute proteins and ancillary factors. Secondary piRNA biogenesis is tightly coupled to transposon repression. It requires processing of the 3-prime end of pre-piRNA during an amplification loop by an as yet unidentified endonuclease. Here, using crystallography, and biochemical assays, we discover that the Drosophila Qin protein, which is a critical member of the core amplification complex, has endonuclease activity. Qin contains five extended Tudor domains, which had been proposed to recognize methylated ligands. Instead, we show that these domains act as RNA-specific nucleases. This supports a role for Qin in the 3-prime end processing of Ago3-bound pre-piRNAs. Extended Tudor domains are frequent in piRNA-processing proteins, suggesting that the uncovered nuclease activity of this protein fold may be key to understanding the piRNA biogenesis.

scholarly journals The core Cas1 protein of CRISPR-Cas I-B in Leptospira shows metal-tunable nuclease activity

2021 ◽  
pp. 100059
Author(s):  
Bhuvan Dixit ◽  
Aman Prakash ◽  
Pankaj Kumar ◽  
Prerana Gogoi ◽  
Manish Kumar
Keyword(s):  
Nuclease Activity ◽  
The Core

scholarly journals Genetic Separation of Sae2 Nuclease Activity from Mre11 Nuclease Functions in Budding Yeast

2017 ◽  
Vol 37 (24) ◽  
Author(s):  
Sucheta Arora ◽  
Rajashree A. Deshpande ◽  
Martin Budd ◽  
Judy Campbell ◽  
America Revere ◽  
...  
Keyword(s):  
Nuclease Activity ◽  
Double Strand Breaks ◽  
Endonuclease Activity ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Dna Ends

ABSTRACT Sae2 promotes the repair of DNA double-strand breaks in Saccharomyces cerevisiae. The role of Sae2 is linked to the Mre11/Rad50/Xrs2 (MRX) complex, which is important for the processing of DNA ends into single-stranded substrates for homologous recombination. Sae2 has intrinsic endonuclease activity, but the role of this activity has not been assessed independently from its functions in promoting Mre11 nuclease activity. Here we identify and characterize separation-of-function mutants that lack intrinsic nuclease activity or the ability to promote Mre11 endonucleolytic activity. We find that the ability of Sae2 to promote MRX nuclease functions is important for DNA damage survival, particularly in the absence of Dna2 nuclease activity. In contrast, Sae2 nuclease activity is essential for DNA repair when the Mre11 nuclease is compromised. Resection of DNA breaks is impaired when either Sae2 activity is blocked, suggesting roles for both Mre11 and Sae2 nuclease activities in promoting the processing of DNA ends in vivo. Finally, both activities of Sae2 are important for sporulation, indicating that the processing of meiotic breaks requires both Mre11 and Sae2 nuclease activities.

scholarly journals The stepwise endonuclease activity of a thermophilic Argonaute protein

2019 ◽  
Author(s):  
Guanhua Xun ◽  
Qian Liu ◽  
Yuesheng Chong ◽  
Zhonglei Li ◽  
Xiang Guo ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Dna Cleavage ◽  
Pyrococcus Furiosus ◽  
Dna Amplification ◽  
Endonuclease Activity ◽  
Gene Detection ◽  
Complementary Sequences ◽  
Argonaute Proteins ◽  
Target Dna ◽  
Single Reaction

AbstractThermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5’-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the PfAgo stepwise endonuclease activity followed by target DNA amplification, we establish a rapid and specific platform for the unambiguously multiplex gene detection, termed RADAR (Renewed-gDNA Assisted DNA-cleavage by Argonaute). In the end, RADAR was applied to distinguish of human papillomavirus of serotypes in patient samples in a single reaction, suggesting that our technique would be adopted for diagnosing application.

Specific inhibition by ATP and other properties of an endonuclease of Neurospora crassa

1968 ◽  
Vol 46 (10) ◽  
pp. 1285-1291 ◽  
Author(s):  
E. Z. Rabin ◽  
M. Mustard ◽  
M. J. Fraser
Keyword(s):  
Neurospora Crassa ◽  
Nuclease Activity ◽  
Enzyme Concentration ◽  
Specific Inhibition ◽  
Endonuclease Activity ◽  
Dna And Rna ◽  
Period 2 ◽  
Lag Period ◽  
Ageing Period ◽  
Soluble Material

An endonuclease specific for single-stranded DNA and RNA was purified from Neurospora crassa conidia by the method of Linn and Lehman (J. Biol. Chem. 240, 1287 (1965)). The activity of the enzyme was measured by following the rate of release of acid-soluble material at 37 °C from either denatured (single-stranded) or native DNA. After an initial lag period, the length of which was inversely proportional to enzyme concentration, the release of acid-soluble material occurred at a rate which was directly proportional to enzyme concentration. Freshly purified enzyme catalyzed the release of acid-soluble material from denatured DNA 50–70 times faster than from native DNA. This ratio of activities increased to approximately 200 following storage at 0–4 °C. Throughout this "ageing" period 2 × 10−4 M ATP inhibited the activity of the enzyme toward both denatured and native DNA by 50%. The following nucleoside phosphates, at a concentration of 4 × 10−4 M, had no effect on the activity of the endonuclease toward denatured DNA: ADP, AMP, ITP, XTP, CTP, dTTP, and UTP. GTP at 4 × 10−4 M inhibited this activity by 15%. Both ATP and dATP at 1 × 10−4 M inhibited endonuclease activity against denatured DNA by about 25%. The inhibition by ATP was noncompetitive over the range of concentration 0.11–0.75 mg DNA per milliliter. Specific inhibition of nuclease activity by ATP has not been previously reported.Several other properties of the endonuclease were examined. There is evidence that endonuclease action was inhibited by accumulation of the products of digestion. The enzyme lost activity toward denatured DNA in a medium containing 0.3 M NaCl but activity was partially restored in the presence of 4 × 10−3 M mercaptoethanol. The endonuclease did not digest DNA–RNA hybrid to any appreciable extent.

Does Decision Making Make a Difference? Patterns of Change Within Canadian National Sport Organizations

1995 ◽  
Vol 9 (3) ◽  
pp. 273-299 ◽  
Author(s):  
Lisa M. Kikulis ◽  
Trevor Slack ◽  
Bob Hinings
Keyword(s):  
Decision Making ◽  
Organizational Design ◽  
Professional Staff ◽  
Sport Organizations ◽  
Strategic Decisions ◽  
Fine Grained ◽  
The Core ◽  
Amateur Sport ◽  
Tightly Coupled ◽  
Dominant Paradigm

The theoretical rationale underpinning this study was that decision making structures are tightly coupled to the core values of organizations and thus have a high impact on organizational design change. Taking a fine-grained approach to the analysis of decision making, the purpose of this study was to determine whether amateur sport organizations have shifted away from the dominant paradigm of volunteer-led decision making in favor of professional staff authority and autonomy over strategic decisions. Data from a population of 36 Canadian national sport organizations were used to show that changes in decision making have occurred. However, the shift in control from volunteers to professionals has not been established. In addition, change in decision making varied according to the direction of change, the decision making dimension, and the decision topic.

scholarly journals Recombinant Thermostable AP Exonuclease from Thermoanaerobacter tengcongensis: Cloning, Expression, Purification, Properties and PCR Application

2013 ◽  
Vol 62 (2) ◽  
pp. 121-129
Author(s):  
SŁAWOMIR DĄBROWSKI ◽  
ANNA BRILLOWSKA-DABROWSKA ◽  
BIRGITTE K. AHRING
Keyword(s):  
Escherichia Coli ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Nuclease Activity ◽  
Protein Product ◽  
Endonuclease Activity ◽  
Gene Encoding ◽  
High Identity ◽  
Thermoanaerobacter Tengcongensis ◽  
Ap Sites

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease III - 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. The very high expression level (57% of total soluble proteins) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg (14000 kU) of pure His6-tagged Tte AP (153 kU/mg) from 1 liter of culture. The optimal conditions of Tte AP endo-, exonuclease and 3'-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. Optimal Tte AP endonuclease activity was observed at 70-75 degrees C, pH 8.0 and at low Mg2+ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3'-5' exonuclease activity and at Mg2+ concentration > 2.0 mM 3' nuclease activity was observed. Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long DNA templates. Tte AP exonuclease eliminated AP-sites in DNA template and improved the efficiency of DNA amplification.

scholarly journals Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating

2020 ◽  
Author(s):  
Anastasia Selyutina ◽  
Mirjana Persaud ◽  
Kyeongeun Lee ◽  
Vineet KewalRamani ◽  
Felipe Diaz-Griffero
Keyword(s):  
Reverse Transcription ◽  
Nuclear Import ◽  
Viral Genome ◽  
Cellular Compartment ◽  
Nuclear Compartment ◽  
Nuclear Compartments ◽  
The Core ◽  
Biochemical Assays ◽  
Infected Cells ◽  
Hiv 1

SUMMARYHIV-1 particles contain a core formed by ~1500 capsid protein monomers housing viral RNA. HIV-1 core uncoating---disassembly---is required for infection. HIV-1 reverse transcription (RT) occurs before or during uncoating, but the cellular compartment where RT and uncoating occurs is unknown. Using imaging and biochemical assays to track HIV-1 capsids in nuclei during infection, we demonstrated that higher-order capsid complexes or complete cores containing viral genome are imported into nuclear compartments. Additionally, inhibition of RT that stabilizes the core during infection does not prevent capsid nuclear import; thus, RT may occur in nuclear compartments. We separated infected cells into cytosolic and nuclear fractions to measure RT during infection. Most observable RT intermediates were enriched in nuclear fractions, suggesting that most HIV-1 RT occurs in the nuclear compartment alongside uncoating. Thus, nuclear import precedes RT and uncoating, fundamentally changing our understanding of HIV-1 infection.

Compensatory coevolution

2019 ◽  
pp. 49-76
Author(s):  
Geoffrey E. Hill
Keyword(s):  
Sexual Selection ◽  
Electron Transport ◽  
Mitochondrial Genes ◽  
Nuclear Genes ◽  
Electron Transport System ◽  
Deleterious Mutations ◽  
The Core ◽  
Slightly Deleterious Mutations ◽  
The Many ◽  
And Control

In most eukaryotes, mitochondrial genes mutate at a higher rate than nuclear genes. In addition, mitochondrial genes are transmitted without recombination, so slightly deleterious mutations are predicted to perpetually accumulate in mt genes. Accumulation of deleterious mutations in mt genes can potentially lead to loss of mitonuclear coadaptation. There is growing evidence that variant nuclear genes evolve so as to compensate for mitochondrial mutations and restore mitochondrial function. This is compensatory coevolution and is the focus of this chapter. The chapter also explores the idea that the many nuclear-encoded subunits in eukaryotic electron transport system enzymes were recruited to regulate and control the core catalytic reactions undertaken by the core mitochondrial subunits. It will also consider the evidence that when mutational erosion occurs, corrupted mitochondrial genotypes can be rescued by introgression of entire mitochondrial genomes. Compensatory coevolution has important implications for speciation, sexual selection, and adaptation.

scholarly journals Piecemeal Microautophagy of the Nucleus Requires the Core Macroautophagy Genes

2008 ◽  
Vol 19 (10) ◽  
pp. 4492-4505 ◽  
Author(s):  
R. Krick ◽  
Y. Muehe ◽  
T. Prick ◽  
S. Bremer ◽  
P. Schlotterhose ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Fusion Genes ◽  
Vacuole Membrane ◽  
The Core ◽  
Atg Proteins ◽  
Biochemical Assays ◽  
Atg Genes ◽  
Mutant Cells ◽  
Specific Membrane

Autophagy is a diverse family of processes that transport cytoplasm and organelles into the lysosome/vacuole lumen for degradation. During macroautophagy cargo is packaged in autophagosomes that fuse with the lysosome/vacuole. During microautophagy cargo is directly engulfed by the lysosome/vacuole membrane. Piecemeal microautophagy of the nucleus (PMN) occurs in Saccharomyces cerevisiae at nucleus-vacuole (NV) junctions and results in the pinching-off and release into the vacuole of nonessential portions of the nucleus. Previous studies concluded macroautophagy ATG genes are not absolutely required for PMN. Here we report using two biochemical assays that PMN is efficiently inhibited in atg mutant cells: PMN blebs are produced, but vesicles are rarely released into the vacuole lumen. Electron microscopy of arrested PMN structures in atg7, atg8, and atg9 mutant cells suggests that NV-junction–associated micronuclei may normally be released from the nucleus before their complete enclosure by the vacuole membrane. In this regard PMN is similar to the microautophagy of peroxisomes (micropexophagy), where the side of the peroxisome opposite the engulfing vacuole is capped by a structure called the “micropexophagy-specific membrane apparatus” (MIPA). The MIPA contains Atg proteins and facilitates terminal enclosure and fusion steps. PMN does not require the complete vacuole homotypic fusion genes. We conclude that a spectrum of ATG genes is required for the terminal vacuole enclosure and fusion stages of PMN.

Sign in / Sign up

Export Citation Format

Share Document