Asymmetric ATP Binding and Hydrolysis Activity of the \u3cem\u3eThermus aquaticus\u3c/em\u3e MutS Dimer Is Key to Modulation of Its Interactions with Mismatched DNA

Prokaryotic MutS and eukaryotic Msh proteins recognize base pair mismatches and insertions or deletions in DNA and initiate mismatch repair. These proteins function as dimers (and perhaps higher order oligomers) and possess an ATPase activity that is essential for DNA repair. Previous studies of Escherichia coli MutS and eukaryotic Msh2−Msh6 proteins have revealed asymmetry within the dimer with respect to both DNA binding and ATPase activities. We have found the Thermus aquaticus MutS protein amenable to detailed investigation of the nature and role of this asymmetry. Here, we show that (a) in a MutS dimer one subunit (S1) binds nucleotide with high affinity and the other (S2) with 10-fold weaker affinity, (b) S1 hydrolyzes ATP rapidly while S2 hydrolyzes ATP at a 30−50-fold slower rate, (c) mismatched DNA binding to MutS inhibits ATP hydrolysis at S1 but slow hydrolysis continues at S2, and (d) interaction between mismatched DNA and MutS is weakened when both subunits are occupied by ATP but remains stable when S1 is occupied by ATP and S2 by ADP. These results reveal key MutS species in the ATPase pathway; S1ADP−S2ATP is formed preferentially in the absence of DNA or in the presence of fully matched DNA, while S1ATP−S2ATP and S1ATP−S2ADP are formed preferentially in the presence of mismatched DNA. These MutS species exhibit differences in interaction with mismatched DNA that are likely important for the mechanism of MutS action in DNA repair

Mismatch Recognition-Coupled Stabilization of Msh2-Msh6 in an ATP-Bound State at the Initiation of DNA Repair

Mismatch repair proteins correct errors in DNA via an ATP-driven process. In eukaryotes, the Msh2-Msh6 complex recognizes base pair mismatches and small insertion/deletions in DNA and initiates repair. Both Msh2 and Msh6 proteins contain Walker ATP-binding motifs that are necessary for repair activity. To understand how these proteins couple ATP binding and hydrolysis to DNA binding/mismatch recognition, the ATPase activity of Saccharomyces cerevisiae Msh2-Msh6 was examined under pre-steady-state conditions. Acid-quench experiments revealed that in the absence of DNA, Msh2-Msh6 hydrolyzes ATP rapidly (burst rate = 3 s-1 at 20 °C) and then undergoes a slow step in the pathway that limits catalytic turnover (kcat = 0.1 s-1). ATP is hydrolyzed similarly in the presence of fully matched duplex DNA; however, in the presence of a G:T mismatch or +T insertion-containing DNA, ATP hydrolysis is severely suppressed (rate = 0.1 s-1). Pulse-chase experiments revealed that Msh2-Msh6 binds ATP rapidly in the absence or in the presence of DNA (rate = 0.1 μM-1 s-1), indicating that for the Msh2-Msh6·mismatched DNA complex, a step after ATP binding but before or at ATP hydrolysis is the rate-limiting step in the pathway. Thus, mismatch recognition is coupled to a dramatic increase in the residence time of ATP on Msh2-Msh6. This mismatch-induced, stable ATP-bound state of Msh2-Msh6 likely signals downstream events in the repair pathway

Role of a Conserved Glutamate Residue in the \u3cem\u3eEscherichia coli\u3c/em\u3e SecA ATPase Mechanism

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis

Impact of Immigration on Native and Ethnic Consumer Identity via Body Image

This research focuses on consumer identity of two under-researched but growing immigrant communities in Australia via the lens of the body image construct. Consistent with an emerging stream of research, body image is viewed as a part of identity. Given the variety of goods and services that have an impact on consumers’ perceptions of their body, and because consumers use products to create and convey desired identities, body image is also viewed as a part of consumer identity. Considering literature on identity, body image, and acculturation, exploratory research was undertaken to determine the impact of immigration on the identities of both immigrants and natives. Specifically, focus groups were conducted on two generations of Filipino- and Indian-Australian women as well as Anglo-Australian women. It was found that second generation immigrants have dual consumer identities where they balance the values, attitudes and lifestyles of both their home (i.e., native or heritage) and host cultures whereas first generation immigrants tend to retain their native consumer identity even if they appear to adopt values, attitudes, and lifestyles of the host culture. The impact of immigrants on consumer identities of native residents who are typically in the majority (i.e., the Anglo group) was not evident. Theoretical and practical implications including recommendations for marketing practitioners are then discussed followed by suggestions for future research

Could NICE guidance on the choice of blood pressure lowering drugs be simplified?

Reecha Sofat and colleagues argue that prescribing advice needs updating in the light of recent evidence that all classes of blood pressure lowering drugs are broadly equivalen

Contribution of Msh2 and Msh6 Subunits to the Asymmetric ATPase and DNA Mismatch Binding Activities of \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e Msh2–Msh6 Mismatch Repair Protein

Previous analyses of both Thermus aquaticus MutS homodimer and Saccharomyces cerevisiae Msh2–Msh6 heterodimer have revealed that the subunits in these protein complexes bind and hydrolyze ATP asymmetrically, emulating their asymmetric DNA binding properties. In the MutS homodimer, one subunit (S1) binds ATP with high affinity and hydrolyzes it rapidly, while the other subunit (S2) binds ATP with lower affinity and hydrolyzes it at an apparently slower rate. Interaction of MutS with mismatched DNA results in suppression of ATP hydrolysis at S1—but which of these subunits, S1 or S2, makes specific contact with the mismatch (e.g., base stacking by a conserved phenylalanine residue) remains unknown. In order to answer this question and to clarify the links between the DNA binding and ATPase activities of each subunit in the dimer, we made mutations in the ATPase sites of Msh2 and Msh6 and assessed their impact on the activity of the Msh2–Msh6 heterodimer (in Msh2–Msh6, only Msh6 makes base specific contact with the mismatch). The key findings are: (a) Msh6 hydrolyzes ATP rapidly, and thus resembles the S1 subunit of the MutS homodimer, (b) Msh2 hydrolyzes ATP at a slower rate, and thus resembles the S2 subunit of MutS, (c) though itself an apparently weak ATPase, Msh2 has a strong influence on the ATPase activity of Msh6, (d) Msh6 binding to mismatched DNA results in suppression of rapid ATP hydrolysis, revealing a “cis” linkage between its mismatch recognition and ATPase activities, (e) the resultant Msh2–Msh6 complex, with both subunits in the ATP-bound state, exhibits altered interactions with the mismatch

Unraveling the directional link between adiposity and inflammation: a bidirectional mendelian randomization approach

<b>Context</b>: Associations between adiposity and circulating inflammation markers are assumed to be causal, although the direction of the relationship has not been proven. <b>Objective</b>: The aim of the study was to explore the causal direction of the relationship between adiposity and inflammation using a bidirectional Mendelian randomization approach. <b>Methods</b>: In the PROSPER study of 5804 elderly patients, we related C-reactive protein (CRP) single nucleotide polymorphisms (SNPs) (rs1800947 and rs1205) and adiposity SNPs (FTO and MC4R) to body mass index (BMI) as well as circulating levels of CRP and leptin. We gave each individual two allele scores ranging from zero to 4, counting each pair of alleles related to CRP levels or BMI. <b>Results</b>: With increasing CRP allele score, there was a stepwise decrease in CRP levels (P for trend < 0.0001) and a 1.98 mg/liter difference between extremes of the allele score distribution, but there was no associated change in BMI or leptin levels (P ≥ 0.89). By contrast, adiposity allele score was associated with 1) an increase in BMI (1.2 kg/m2 difference between extremes; P for trend 0.002); 2) an increase in circulating leptin (5.77 ng/ml difference between extremes; P for trend 0.0027); and 3) increased CRP levels (1.24 mg/liter difference between extremes; P for trend 0.002). <b>Conclusions</b>: Greater adiposity conferred by FTO and MC4R SNPs led to higher CRP levels, with no evidence for any reverse pathway. Future studies should extend our findings to other circulating inflammatory parameters. This study illustrates the potential power of Mendelian randomization to dissect directions of causality between intercorrelated metabolic factors

Overproduction and Analysis of Eukaryotic Multiprotein Complexes in \u3cem\u3eEscherichia coli\u3c/em\u3e Using a Dual-vector Strategy

Biochemical studies of eukaryotic proteins are often constrained by low availability of these typically large, multicomponent protein complexes in pure form. Escherichia coli is a commonly used host for large-scale protein production; however, its utility for eukaryotic protein production is limited because of problems associated with transcription, translation, and proper folding of proteins. Here we describe the development and testing of pLANT, a vector that addresses many of these problems simultaneously. The pLANT vector contains a T7 promoter-controlled expression unit, a p15A origin of replication, and genes for rare transfer RNAs and kanamycin resistance. Thus, the pLANT vector can be used in combination with the pET vector to coexpress multiple proteins in E. coli. Using this approach, we have successfully produced high-milligram quantities of two different Saccharomyces cerevisiae complexes in E. coli: the heterodimeric Msh2–Msh6 mismatch repair protein (248 kDa) and the five-subunit replication factor C clamp loader (250 kDa). Quantitative analyses indicate that these proteins are fully active, affirming the utility of pLANT+pET-based production of eukaryotic proteins in E. coli for in vitro studies of their structure and function

Voltage Stability Analysis of Grid-Connected Wind Farms with FACTS: Static and Dynamic Analysis

Recently, analysis of some major blackouts and failures of power system shows that voltage instability problem has been one of the main reasons of these disturbances and networks collapse. In this paper, a systematic approach to voltage stability analysis using various techniques for the IEEE 14-bus case study, is presented. Static analysis is used to analyze the voltage stability of the system under study, whilst the dynamic analysis is used to evaluate the performance of compensators. The static techniques used are Power Flow, V–P curve analysis, and Q–V modal analysis. In this study, Flexible Alternating Current Transmission system (FACTS) devices- namely, Static Synchronous Compensators (STATCOMs) and Static Var Compensators (SVCs) - are used as reactive power compensators, taking into account maintaining the violated voltage magnitudes of the weak buses within the acceptable limits defined in ANSI C84.1. Simulation results validate that both the STATCOMs and the SVCs can be effectively used to enhance the static voltage stability and increasing network loadability margin. Additionally, based on the dynamic analysis results, it has been shown that STATCOMs have superior performance, in dynamic voltage stability enhancement, compared to SVCs

Challenges in the management of pediatric blepharokeratoconjunctivis / ocular rosacea

Introduction: Childhood blepharokeratoconjunctivitis is a common lid margin inflammation with secondary ocular surface disease. Its etiology is unclear and there are no randomized controlled trials to support the superiority of any treatment option. / Areas covered: We searched the following databases; Cochrane Central Register of Controlled Trials, Ovid MEDLINE and affiliated Ovid databases, EMBASE, the ISRCTN registry, Clinical- Trials.gov and the World Health Organization International Clinical Trials Registry Platform. Due to the paucity of pediatric data we also considered information from articles focused on adults. / Expert commentary: Treatment is based on the assumption that the mechanisms of BKC and rosacea keratitis are the same: meibomian gland dysfunction, bacterial colonisation of the lid margin, delayed type hypersensitivity, Demodex folliculorum, genetic predisposition and Toll-like receptors inducing release of pro-inflammatory cytokines. Generally accepted grading scales are needed. Randomized clinical trials are needed to evaluate treatment options. The effects of antibiotics, immunomodulators, osmoprotectants and essential fatty acids need further investigation