scholarly journals Structure and function of a novel endonuclease acting on branched DNA substrates

2011 ◽  
Vol 39 (1) ◽  
pp. 145-149 ◽  
Author(s):  
Christophe Creze ◽  
Roxane Lestini ◽  
Joelle Kühn ◽  
Alessio Ligabue ◽  
Hubert F. Becker ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Nuclease Activity ◽  
Nuclear Antigen ◽  
Double Stranded Dna ◽  
Dna Junctions ◽  
Branched Dna ◽  
And Function ◽  
Proliferating Cell ◽  
Pyrococcus Abyssi

Branched DNA structures that occur during DNA repair and recombination must be efficiently processed by structure-specific endonucleases in order to avoid cell death. In the present paper, we summarize our screen for new interaction partners for the archaeal replication clamp that led to the functional characterization of a novel endonuclease family, dubbed NucS. Structural analyses of Pyrococcus abyssi NucS revealed an unexpected binding site for ssDNA (single-stranded DNA) that directs, together with the replication clamp, the nuclease activity of this protein towards ssDNA–dsDNA (double-stranded DNA) junctions. Our studies suggest that understanding the detailed architecture and dynamic behaviour of the NucS (nuclease specific for ssDNA)–PCNA (proliferating-cell nuclear antigen) complex with DNA will be crucial for identification of its physiologically relevant activities.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
Author(s):  
Marie Anne Richard ◽  
Hannah Pallubinsky ◽  
Denis P. Blondin
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Functional Characterization ◽  
Human Infants ◽  
Positron Emission ◽  
Brown Adipose ◽  
Treatment Of Obesity ◽  
And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

Functional characterization of the trypanosome translational repressor SCD6

2013 ◽  
Vol 457 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Marina Cristodero ◽  
Bernd Schimanski ◽  
Manfred Heller ◽  
Isabel Roditi
Keyword(s):  
Functional Characterization ◽  
Stress Granule ◽  
Granule Formation ◽  
Translational Repressor ◽  
And Function

Trypanosomal SCD6 is a general repressor of translation. It is not required for stress granule formation and, unusually, does not interact with the helicase DHH1. We analysed domains involved in the localization and function of TbSCD6 and identified interacting partners.

scholarly journals K6-linked SUMOylation of BAF regulates nuclear integrity and DNA replication in mammalian cells

2020 ◽  
Vol 117 (19) ◽  
pp. 10378-10387 ◽  
Author(s):  
Qiaoyu Lin ◽  
Bin Yu ◽  
Xiangyang Wang ◽  
Shicong Zhu ◽  
Gan Zhao ◽  
...  
Keyword(s):  
Dna Replication ◽  
Nuclear Localization ◽  
Proliferating Cell Nuclear Antigen ◽  
Mammalian Cells ◽  
Regulatory Mechanism ◽  
Nuclear Antigen ◽  
Multiple Functions ◽  
And Function ◽  
Proliferating Cell ◽  
Replication Failure

Barrier-to-autointegration factor (BAF) is a highly conserved protein in metazoans that has multiple functions during the cell cycle. We found that BAF is SUMOylated at K6, and that this modification is essential for its nuclear localization and function, including nuclear integrity maintenance and DNA replication. K6-linked SUMOylation of BAF promotes binding and interaction with lamin A/C to regulate nuclear integrity. K6-linked SUMOylation of BAF also supports BAF binding to DNA and proliferating cell nuclear antigen and regulates DNA replication. SENP1 and SENP2 catalyze the de-SUMOylation of BAF at K6. Disrupting the SUMOylation and de-SUMOylation cycle of BAF at K6 not only disturbs nuclear integrity, but also induces DNA replication failure. Taken together, our findings demonstrate that SUMOylation at K6 is an important regulatory mechanism that governs the nuclear functions of BAF in mammalian cells.

scholarly journals Structural and Functional Characterization of RecG Helicase under Dilute and Molecular Crowding Conditions

2012 ◽  
Vol 2012 ◽  
pp. 1-8
Author(s):  
Sarika Saxena ◽  
Satoru Nagatoishi ◽  
Daisuke Miyoshi ◽  
Naoki Sugimoto
Keyword(s):  
Functional Characterization ◽  
Cd Spectroscopy ◽  
Helical Conformation ◽  
Atp Binding ◽  
Molecular Crowding ◽  
Active Structure ◽  
Dna Junctions ◽  
Unique Protein

In an ATP-dependent reaction, theEscherichia coliRecG helicase unwinds DNA junctionsin vitro. We present evidence of a unique protein conformational change in the RecG helicase from anα-helix to aβ-strand upon an ATP binding under dilute conditions using circular dichroism (CD) spectroscopy. In contrast, under molecular crowding conditions, theα-helical conformation was stable even upon an ATP binding. These distinct conformational behaviors were observed to be independent of Na+and Mg2+. Interestingly, CD measurements demonstrated that the spectra of a frayed duplex decreased with increasing of the RecG concentration both under dilute and molecular crowding conditions in the presence of ATP, suggesting that RecG unwound the frayed duplex. Our findings raise the possibility that theα-helix andβ-strand forms of RecG are a preactive and an active structure with the helicase activity, respectively.

Characterization of plant XRCC1 and its interaction with proliferating cell nuclear antigen

Planta ◽  
2008 ◽  
Vol 227 (6) ◽  
pp. 1233-1241 ◽  
Author(s):  
Yukinobu Uchiyama ◽  
Yuko Suzuki ◽  
Kengo Sakaguchi
Keyword(s):  
Proliferating Cell Nuclear Antigen ◽  
Nuclear Antigen ◽  
Proliferating Cell

scholarly journals Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus

2021 ◽  
Author(s):  
Leonhard M Kick ◽  
Marie-Kristin von Wrisberg ◽  
Leander S Runtsch ◽  
Sabine Schneider
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Rhodobacter Capsulatus ◽  
Purple Bacteria ◽  
Functional Characterization ◽  
Regulation Of Gene Expression ◽  
Nuclease Activity ◽  
Site Directed Mutagenesis ◽  
And Function ◽  
Key Residues

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key-residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

scholarly journals Ontogenic, phenotypic, and functional characterization of XCR1+ dendritic cells leads to a consistent classification of intestinal dendritic cells based on the expression of XCR1 and SIRPα

2014 ◽  
Author(s):  
Martina Becker ◽  
Steffen Güttler ◽  
Annabell Bachem ◽  
Evelyn Hartung ◽  
Ahmed Mora ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
T Cell ◽  
Functional Characterization ◽  
Cross Presentation ◽  
Intestinal Immune System ◽  
Lineage Marker ◽  
And Function ◽  
First Time

In the past, lack of lineage markers confounded the classification of dendritic cells (DC) in the intestine and impeded a full understanding of their location and function. We have recently shown that the chemokine receptor XCR1 is a lineage marker for cross-presenting DC in the spleen. Now we provide evidence that intestinal XCR1+ DC largely, but not fully, overlap with CD103+ CD11b- DC, the hypothesized correlate of “cross-presenting DC” in the intestine, and are selectively dependent in their development on the transcription factor Batf3. XCR1+ DC are located in the villi and epithelial crypts of the lamina propria of the small intestine, the T cell zones of Peyer’s Patches, and in the T cell zones and sinuses of the draining mesenteric lymph node. Functionally, we could demonstrate for the first time that XCR1+ / CD103+ CD11b- DC excel in the cross-presentation of orally applied antigen. Together, our data show that XCR1 is a lineage marker for cross-presenting DC also in the intestinal immune system. Further, extensive phenotypic analyses reveal that expression of the integrin SIRPα consistently demarcates the XCR1- DC population. We propose a simplified and consistent classification system for intestinal DC based on the expression of XCR1 and SIRPα.

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