Differential functions of FANCI and FANCD2 ubiquitination stabilize ID2 complex on DNA
The Fanconi anaemia (FA) pathway is a dedicated pathway for the repair of DNA interstrand crosslinks and is additionally activated in response to other forms of replication stress. A key step in the FA pathway is the monoubiquitination of each of the two subunits (FANCI and FANCD2) of the ID2 complex on specific lysine residues.
However, the molecular function of these modifications has been unknown for nearly two decades. Here, we find that ubiquitination of FANCD2 acts to increase ID2’s affinity for double-stranded DNA via promoting a large-scale conformational change in the complex. The resulting complex encircles DNA, by forming a secondary “Arm” ID2 interface.
Ubiquitination of FANCI, on the other hand, largely protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI’s ubiquitin being important for this protection. In effect, both of these post-translational modifications function to stabilize a conformation in which the ID2 complex encircles DNA.
Keywords: DNA repair; Fanconi anaemia; deubiquitination; protein-DNA interactions; ubiquitin
Description: This antibody is specific for serotype 5C and does not react with other species. Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. It is an opportunistic pathogen of plants and humans and can infect the urinary tract, respiratory and gastrointestinal system, soft tissues, bones and joints leading to severe systemic infections of immunosuppressed patients in hospitals. P. aeruginosa secretes a variety of pigments, including pyocyanin (blue-green), fluorescein (pyoverdin), and pyorubin (red-brown). This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and, in its absence, it is also able to ferment arginine by substrate-level phosphorylation. Adaptation to microaerobic or anaerobic environments is essential for certain lifestyles of P. aeruginosa, such as during lung infection in cystic fibrosis patients where thick layers of alginate surrounding bacterial mucoid cells can limit the diffusion of oxygen.
Description: This antibody is specific for serotype 5C and does not react with other species. Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. It is an opportunistic pathogen of plants and humans and can infect the urinary tract, respiratory and gastrointestinal system, soft tissues, bones and joints leading to severe systemic infections of immunosuppressed patients in hospitals. P. aeruginosa secretes a variety of pigments, including pyocyanin (blue-green), fluorescein (pyoverdin), and pyorubin (red-brown). This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and, in its absence, it is also able to ferment arginine by substrate-level phosphorylation. Adaptation to microaerobic or anaerobic environments is essential for certain lifestyles of P. aeruginosa, such as during lung infection in cystic fibrosis patients where thick layers of alginate surrounding bacterial mucoid cells can limit the diffusion of oxygen.
Description: This antibody is specific for serotype 5C and does not react with other species. Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. It is an opportunistic pathogen of plants and humans and can infect the urinary tract, respiratory and gastrointestinal system, soft tissues, bones and joints leading to severe systemic infections of immunosuppressed patients in hospitals. P. aeruginosa secretes a variety of pigments, including pyocyanin (blue-green), fluorescein (pyoverdin), and pyorubin (red-brown). This organism can achieve anaerobic growth with nitrate as a terminal electron acceptor, and, in its absence, it is also able to ferment arginine by substrate-level phosphorylation. Adaptation to microaerobic or anaerobic environments is essential for certain lifestyles of P. aeruginosa, such as during lung infection in cystic fibrosis patients where thick layers of alginate surrounding bacterial mucoid cells can limit the diffusion of oxygen.
Description: This antibody is specific for serotype 6C and does not react with other species. Pseudomonas aeruginosa is Gram-negative, aerobic, rod-shaped bacteria with unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants. P. aeruginosa bacteria are clinically important because they are resistant to most antibiotics and they are capable of surviving in conditions that few other organisms can tolerate. Pseudomonas is often encountered in hospital and clinical work because it is a major cause of hospital acquired (nosocomial) infections. Its main targets are immuno-compromised individuals, burn victims, and individuals on respirators or with indwelling catheters. Additionally, these pathogens colonize the lungs of cystic fibrosis patients. P. aeruginosa is often identified by its pearlescent appearance and grape-like odor in vitro. Definitive clinical identification of P. aeruginosa includes identifying the production of both pyocyanin and fluorescein as well as its ability to grow at 42C. P. aeruginosa is capable of growth in diesel and jet fuel, where it is known as hydrocarbon utilizing microorganisms (or HUM bugs), causing microbial corrosion.
Description: This antibody is specific for serotype 6C and does not react with other species. Pseudomonas aeruginosa is Gram-negative, aerobic, rod-shaped bacteria with unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants. P. aeruginosa bacteria are clinically important because they are resistant to most antibiotics and they are capable of surviving in conditions that few other organisms can tolerate. Pseudomonas is often encountered in hospital and clinical work because it is a major cause of hospital acquired (nosocomial) infections. Its main targets are immuno-compromised individuals, burn victims, and individuals on respirators or with indwelling catheters. Additionally, these pathogens colonize the lungs of cystic fibrosis patients. P. aeruginosa is often identified by its pearlescent appearance and grape-like odor in vitro. Definitive clinical identification of P. aeruginosa includes identifying the production of both pyocyanin and fluorescein as well as its ability to grow at 42C. P. aeruginosa is capable of growth in diesel and jet fuel, where it is known as hydrocarbon utilizing microorganisms (or HUM bugs), causing microbial corrosion.
Description: This antibody is specific for serotype 6C and does not react with other species. Pseudomonas aeruginosa is Gram-negative, aerobic, rod-shaped bacteria with unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an opportunistic pathogen of plants. P. aeruginosa bacteria are clinically important because they are resistant to most antibiotics and they are capable of surviving in conditions that few other organisms can tolerate. Pseudomonas is often encountered in hospital and clinical work because it is a major cause of hospital acquired (nosocomial) infections. Its main targets are immuno-compromised individuals, burn victims, and individuals on respirators or with indwelling catheters. Additionally, these pathogens colonize the lungs of cystic fibrosis patients. P. aeruginosa is often identified by its pearlescent appearance and grape-like odor in vitro. Definitive clinical identification of P. aeruginosa includes identifying the production of both pyocyanin and fluorescein as well as its ability to grow at 42C. P. aeruginosa is capable of growth in diesel and jet fuel, where it is known as hydrocarbon utilizing microorganisms (or HUM bugs), causing microbial corrosion.
Description: Recombinant Streptococcus pyogenes serotype M28 Streptopain(speB) expressed in E.coli
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Fluorometric determination of agrA gene transcription in methicillin-resistant Staphylococcus aureus with a graphene oxide-based assay using strand-displacement polymerization recycling and hybridization chain reaction
A graphene oxide (GO)-based fluorescent bioassay was developed to quantify agrA gene transcription (its mRNA) in methicillin-resistant Staphylococcus aureus (MRSA). This method is based on the use of Klenow fragment (KF)-assisted target recycling amplification and hybridization chain reaction (HCR). A triple complex was designed that contained a capture probe (CP), a trigger probe (TP), and a help probe (HP), which were partially complementary to one another.
In the absence of the target, all the oligonucleotides labeled with carboxyfluorescein (FAM) are adsorbed onto the surface of GO by π-stacking interactions. This adsorption quenches the FAM signal. On the contrary, the target RNA causes the triple complex to disintegrate and initiates strand-displacement polymerization reaction (SDPR) and HCR in the presence of the appropriate raw materials, including the primer, KF, dNTPs, hairpin 1 (H1), and hairpin 2 (H2), generating double-stranded DNA (dsDNA) products.
These dsDNA products are repelled by GO and produce strong fluorescence, measured at excitation/emission wavelengths of 480/514 nm. The fluorescent signal is greatly amplified by SYBR Green I (SGI) due to the synergistic effect of dsDNA-SGI. The target was assayed with this method at concentrations in the range 10 fM to 100 pM, and the detection limit (LOD) was 10 fM.
This method also displayed good applicability in the analysis of real samples. It provides a new way of monitoring biofilm formation and studying the mechanisms of drug actions. Graphical abstract Schematic representation of the graphene oxide-based fluorescent bioassay for agrA gene transcription in methicillin-resistant Staphylococcus aureus by using strand-displacement polymerization recycling and hybridization chain reaction.
The structure of human CST reveals a decameric assembly bound to telomeric DNA
The CTC1-STN1-TEN1 (CST) complex is essential for telomere maintenance and resolution of stalled replication forks genome-wide. Here, we report the 3.0-angstrom cryo-electron microscopy structure of human CST bound to telomeric single-stranded DNA (ssDNA), which assembles as a decameric supercomplex.
The atomic model of the 134-kilodalton CTC1 subunit, built almost entirely de novo, reveals the overall architecture of CST and the DNA-binding anchor site. The carboxyl-terminal domain of STN1 interacts with CTC1 at two separate docking sites, allowing allosteric mediation of CST decamer assembly. Furthermore, ssDNA appears to staple two monomers to nucleate decamer assembly.
CTC1 has stronger structural similarity to Replication Protein A than the expected similarity to yeast Cdc13. The decameric structure suggests that CST can organize ssDNA analogously to the nucleosome’s organization of double-stranded DNA.
