To prevent ATR activation, telomeres deploy the single-stranded DNA binding activity of TPP1/POT1a. POT1a blocks the binding of RPA to telomeres, suggesting that ATR is repressed through RPA ...exclusion. However, comparison of the DNA binding affinities and abundance of TPP1/POT1a and RPA indicates that TPP1/POT1a by itself is unlikely to exclude RPA. We therefore analyzed the central shelterin protein TIN2, which links TPP1/POT1a (and POT1b) to TRF1 and TRF2 on the double-stranded telomeric DNA. Upon TIN2 deletion, telomeres lost TPP1/POT1a, accumulated RPA, elicited an ATR signal, and showed all other phenotypes of POT1a/b deletion. TIN2 also affected the TRF2-dependent repression of ATM kinase signaling but not to TRF2-mediated inhibition of telomere fusions. Thus, while TIN2 has a minor contribution to the repression of ATM by TRF2, its major role is to stabilize TPP1/POT1a on the ss telomeric DNA, thereby allowing effective exclusion of RPA and repression of ATR signaling.
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► Biochemical data do not explain how TPP1/POT1 excludes RPA from single-stranded telomeric DNA ► Deletion of TIN2 shows that TPP1/POT1 depends on TIN2 for telomere binding ► Without the TIN2 tether, TPP1/POT1 fails to block RPA binding and ATR activation ► In addition, TIN2 contributes to TRF2-dependent repression of ATM signaling
The factor that plays the essential role in hydrogen-related failure has been examined for Inconel 625 and iron by means of tensile testing with interposed unloading and reloading with/without ...hydrogen charging. Aging at 30
°C or annealing at 200
°C was conducted during the unloaded stage in order to diffuse out hydrogen or to anneal out strain-induced defects. Hydrogen thermal desorption analysis was used to evaluate strain-induced defects that act as trapping sites of hydrogen. Fracture strain decreased in the initially hydrogen-charged specimens even though hydrogen was absent at the late stage of straining. Annealing at 200
°C at the unloaded stage almost completely recovered the decrease in fracture strain. Enhancement of strain-induced defects by hydrogen and their involvement in degradation were revealed by means of hydrogen thermal desorption analysis. The results provide direct evidence of the primary role of vacancies rather than hydrogen itself in hydrogen degradation, and agree well with the hydrogen-enhanced strain-induced vacancy model with respect to the mechanism of hydrogen-related failure.
Biochemical characterization of each gene product encoded in the genome is essential to understand how cells are regulated. The bottleneck has been and still is in how the gene products can be ...obtained. The wheat cell-free protein synthesis system we have developed is a powerful method for preparation of many different proteins at a time and also for preparation of large amounts of specific proteins for biochemical and structural analyses. Here, we show a method for preparation of the wheat embryo extract useful for the cell-free reactions, by which 5 ml of a high-activity extract is obtained in 4-5 d. We also describe the methods for small- and large-scale protein synthesis by hands-down operations with the use of mRNAs prepared by transcription of PCR products and pEU plasmids harboring the target cDNAs, which need 2-4 d excepting the time required for plasmid preparation.
In Vivo Stoichiometry of Shelterin Components Takai, Kaori K.; Hooper, Sarah; Blackwood, Stephanie ...
Journal of biological chemistry/The Journal of biological chemistry,
01/2010, Volume:
285, Issue:
2
Journal Article
Peer reviewed
Open access
Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used ...quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1·POT1 heterodimer was present 50–100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1·POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ∼10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1·POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.
We report an unexpected role for Tel2 in the expression of all mammalian phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Although Tel2 was identified as a budding yeast gene required ...for the telomere length maintenance, we found no obvious telomeric function for mammalian Tel2. Targeted gene deletion showed that mouse Tel2 is essential in embryonic development, embryonic stem (ES) cells, and embryonic fibroblasts. Conditional deletion of Tel2 from embryonic fibroblasts compromised their response to IR and UV, diminishing the activation of checkpoint kinases and their downstream effectors. The effects of Tel2 deletion correlated with significantly reduced protein levels for the PI3K-related kinases ataxia telangiectasia mutated (ATM), ATM and Rad3 related (ATR), DNA-dependent protein kinase catalytic subunit ataxia (DNA-PKcs). Tel2 deletion also elicited specific depletion of the mammalian target of rapamycin (mTOR), suppressor with morphological effect on genitalia 1 (SMG1), and transformation/transcription domain-associated protein (TRRAP), and curbed mTOR signaling, indicating that Tel2 affects all six mammalian PIKKs. While Tel2 deletion did not alter PIKK mRNA levels, in vivo pulse labeling experiments showed that Tel2 controls the stability of ATM and mTOR. Each of the PIKK family members associated with Tel2 in vivo and in vitro experiments indicated that Tel2 binds to part of the HEAT repeat segments of ATM and mTOR. These data identify Tel2 as a highly conserved regulator of PIKK stability.
Interactions between interior substitutional nitrogen defects and surface unsaturated dangling bonds in synthetic nanodiamonds of ∼25 nm size were explored experimentally and theoretically. The ...experimental results demonstrate the disappearance of the specific paramagnetism of nitrogen centers in the smallest nanoparticles isolated after processing large micron diamonds in a ball mill, accompanied by the formation of unsaturated surface dangling bonds and internal defects. First principles modelling confirms the vanishing of the magnetic moments related with nitrogen centers even for distances from the surface defects greater than 1 nm. To understand this effect, we consider a bond reconstruction scheme with the formation of several carbon-carbon double bonds in the area between the interior and surface point defects. The scheme is in agreement with the changes in electron density through the distance between the two defects. The developed approach can be used to describe the interactions between various defects in carbon-based systems.
Interactions between interior substitutional nitrogen defects and surface unsaturated dangling bonds in synthetic nanodiamonds of ∼25 nm size were explored experimentally and theoretically.
This manuscript reports the analysis of the
G′ band profile in the Raman spectra of nanographites with different degrees of stacking order. Since the
G′ band scattering coming from the 2D and 3D ...phases coexisting in the same sample can be nicely distinguished, the relative volumes of 3D and 2D graphite phases present in the samples can be estimated from their Raman spectra. The comparison between Raman scattering and X-Ray diffraction data shows that Raman spectroscopy can be used as an alternative tool for measuring the degree of stacking order of graphitic systems.
The numbers of lattice defects formed by applying cyclic prestress with/without hydrogen for various numbers of cycles and strain rates during cyclic prestress were compared for tempered martensitic ...steel. A tensile test was also carried out to evaluate hydrogen embrittlement susceptibility following the application of cyclic prestress. The results showed that when cyclic prestress was applied without hydrogen, the number of cycles and strain rate had no apparent effect on mechanical properties and fracture morphology at the time of the subsequent tensile test. In contrast, when cyclic prestress was applied with hydrogen, the fracture strain and fracture stress decreased with an increasing number of prestress cycles and a decreasing strain rate, and the fracture morphology exhibited brittle fracture, signifying an increase in hydrogen embrittlement susceptibility at the time of the tensile test. The number of hydrogen-enhanced lattice defects also increased with increasing number of cycles and a decreasing strain rate was found when cyclic prestress was applied with hydrogen. These results indicate a correlation between hydrogen embrittlement susceptibility and the number of hydrogen-enhanced lattice defects. The kinds of increased hydrogen-enhanced lattice defects were probably vacancies and vacancy clusters formed by the interactions between hydrogen and dislocation movement during the application of cyclic prestress. The vacancies and vacancy clusters formed during the application of cyclic prestress with hydrogen presumably caused intergranular fracture and increased hydrogen embrittlement susceptibility.
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