Biochemical analysis of the N-terminal domain of human RAD54B

The human RAD54B protein is a paralog of the RAD54 protein, which plays important roles in homologous recombination. RAD54B contains an N-terminal region outside the SWI2/SNF2 domain that shares less conservation with the corresponding region in RAD54. The biochemical roles of this region of RAD54B are not known, although the corresponding region in RAD54 is known to physically interact with RAD51. In the present study, we have biochemically characterized an N-terminal fragment of RAD54B, consisting of amino acid residues 26–225 (RAD54B26–225). This fragment formed a stable dimer in solution and bound to branched DNA structures. RAD54B26–225 also interacted with DMC1 in both the presence and absence of DNA. Ten DMC1 segments spanning the entire region of the DMC1 sequence were prepared, and two segments, containing amino acid residues 153–214 and 296–340, were found to directly bind to the N-terminal domain of RAD54B. A structural alignment of DMC1 with the Methanococcus voltae RadA protein, a homolog of DMC1 in the helical filament form, indicated that these RAD54B-binding sites are located near the ATP-binding site at the monomer–monomer interface in the DMC1 helical filament. Thus, RAD54B binding may affect the quaternary structure of DMC1. These observations suggest that the N-terminal domain of RAD54B plays multiple roles of in homologous recombination

DNA end resection by Dna2–Sgs1–RPA and its stimulation by Top3–Rmi1 and Mre11–Rad50–Xrs2

The repair of DNA double-strand breaks (DSBs) by homologous recombination requires processing of broken ends. For repair to start, the DSB must first be resected to generate a 3′-single-stranded DNA (ssDNA) overhang, which becomes a substrate for the DNA strand exchange protein, Rad51 (ref. 1). Genetic studies have implicated a multitude of proteins in the process, including helicases, nucleases and topoisomerases. Here we biochemically reconstitute elements of the resection process and reveal that it requires the nuclease Dna2, the RecQ-family helicase Sgs1 and the ssDNA-binding protein replication protein-A (RPA). We establish that Dna2, Sgs1 and RPA constitute a minimal protein complex capable of DNA resection in vitro. Sgs1 helicase unwinds the DNA to produce an intermediate that is digested by Dna2, and RPA stimulates DNA unwinding by Sgs1 in a species-specific manner. Interestingly, RPA is also required both to direct Dna2 nucleolytic activity to the 5′-terminated strand of the DNA break and to inhibit 3′ to 5′ degradation by Dna2, actions that generate and protect the 3′-ssDNA overhang, respectively. In addition to this core machinery, we establish that both the topoisomerase 3 (Top3) and Rmi1 complex and the Mre11–Rad50–Xrs2 complex (MRX) have important roles as stimulatory components. Stimulation of end resection by the Top3–Rmi1 heterodimer and the MRX proteins is by complex formation with Sgs1 (refs 5, 6), which unexpectedly stimulates DNA unwinding. We suggest that Top3–Rmi1 and MRX are important for recruitment of the Sgs1–Dna2 complex to DSBs. Our experiments provide a mechanistic framework for understanding the initial steps of recombinational DNA repair in eukaryotes

Why do authoritarian regimes provide public goods? Policy communities, external shocks and ideas in China’s rural social policy making

Recent research on authoritarian regimes argues that they provide public goods in order to prevent rebellion. This essay shows that the ‘threat of rebellion’ alone cannot explain Chinese party-state policies to extend public goods to rural residents in the first decade of the twenty-first century. Drawing on theories of policy making, it argues that China’s one-party regime extended public goods to the rural population under the influence of ideas and policy options generated by policy communities of officials, researchers, international organisations and other actors. The party-state centre adopted and implemented these ideas and policy options when they provided solutions to external shocks and supported economic development goals. Explanations of policies and their outcomes in authoritarian political systems need to include not only ‘dictators’ but also other actors, and the ideas they generate

'It would be easier if she’d died’: young people with parents with dementia articulating inadmissible stories

In the U.K. context where the emphasis is (quite rightly) on living well with dementia, on positivity and enabling approaches, it can be difficult for researchers to investigate and report negative experiences. Failing to re-present perceptions and experiences as they are lived, however, does a serious disservice to the research endeavor and can prevent policy and service development and positive change. In this article, we present some stories told by participants in an Alzheimer’s Society (United Kingdom) Funded project uniquely investigating the perceptions and experiences of children and young people who have a parent with dementia. Sometimes the stories were not easy to hear, especially when they challenged dominant master narratives around dementia. We discuss our view that when the young people we spoke with told us how things were for them, we were ethically bound to respect and disseminate their accounts

Environment change, economy change and reducing conflict at source

At a time when fossil fuel burning, nationalism, ethnic and religious intolerance, and other retrograde steps are being promoted, the prospects for world peace and environmental systems stability may appear dim. Yet now is it the more important to continue to examine the sources of conflict. A major obstacle to general progress is the currently dominant economic practice and theory, which is here called the economy-as-usual, or economics-as-usual, as appropriate. A special obstacle to constructive change is the language in which economic matters are usually discussed. This language is narrow, conservative, technical and often obscure. The rapid changes in the environment (physical and living) are largely kept in a separate compartment. If, however, the partition is removed, economics -as-usual, with its dependence on growth and its widening inequality, is seen to be unsustainable. Radical economic change, for better or worse, is to be expected. Such change is here called economy change. The change could be for the better if it involved an expansion of the concept of economics itself, along the lines of oikonomia, a modern revival of a classical Greek term for management or household. In such an expanded view, not everything of economic value can be measured. It is argued that economics-as-usual is the source of much strife. Some features are indicated of a less conflictual economy - more just, cooperative and peaceful. These features include a dignified life available to all people as of right, the word 'wealth' being reconnected with weal, well and well-being, and 'work' being understood as including all useful activity

Chemical Components from the Light Petroleum Soluble Fraction of Uvaria cordata (Dunal) Alston

Chromatographic separation of the light petroleum extract from the stem bark of Uvaria cordata (Dunal) Alston led to the isolation of the triterpenoids glutinol and taraxerol in addition to the cyclohexene derivatives, pipoxide and its chlorohydrin. A small amount of benzyl benzoate was also isolated

A mutation in the melon Vacuolar Protein Sorting 41prevents systemic infection of Cucumber mosaic virus

[EN] In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem.The TILLING platform is supported by the Program Saclay Plant Sciences (SPS, ANR-10-LABX-40) and the European Research Council (ERC-SEXYPARTH). This work was supported by grants AGL2009-12698-C02-01 and AGL2012-40130-C02-01 from the Spanish Ministry of Science and Innovation, the Spanish Ministry of Econom and Competitiveness, through the "Severo Ochoa Programme for Centres of Excellence in R&D" 2016-2019 (SEV-2015-0533)" and the CERCA Programme/Generalitat de Catalunya.Giner, A.; Pascual, L.; Bourgeois, M.; Gyetvai, G.; Rios, P.; Picó Sirvent, MB.; Troadec, C.... (2017). 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On becoming (un)committed: A taxonomy and test of newcomer on-boarding scenarios

How does the bond between the newcomer and the organization develop over time? Process research on temporal patterns of newcomer's early commitment formation has been very scarce because theory and appropriate longitudinal research designs in this area are lacking. From extant research we extract three process-theoretical accounts regarding how the newcomer adjustment process evolves over time: (1) Learning to Love; (2) Honeymoon Hangover; and (3) High Match, Moderate Match, or Low Match. From these scenarios we develop a taxonomy of newcomer adjustment scenarios. Further, we empirically verify these different scenarios by examining naturally occurring "trajectory classes," which are found to display strengthening, weakening, or stabilizing of the employee-organization linkage. For this, we use a sample of 72 Ph. D. graduates whose organizational commitment history was recorded in their first 25 consecutive weeks of new employment. In closing, we discuss the theoretical and practical implications of the scenario-based approach

The wonders of flap endonucleases: structure, function, mechanism and regulation.

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication