Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3

double Holliday junction (dHJ) is a central intermediate of homologous recombination that can be processed to yield crossover or non-crossover recombination products. To preserve genomic integrity, cells possess mechanisms to avoid crossing over. We show that Saccharomyces cerevisiae Sgs1 and Top3 proteins are sufficient to migrate and disentangle a dHJ to produce exclusively non-crossover recombination products, in a reaction termed "dissolution." We show that Rmi1 stimulates dHJ dissolution at low Sgs1-Top3 protein concentrations, although it has no effect on the initial rate of Holliday junction (HJ) migration. Rmi1 serves to stimulate DNA decatenation, removing the last linkages between the repaired and template DNA molecules. Dissolution of a dHJ is a highly efficient and concerted alternative to nucleolytic resolution that prevents crossing over of chromosomes during recombinational DNA repair in mitotic cells and thereby contributes to genomic integrity

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

The ethics of biobanking: key issues and controversies

The ethics of biobanking is one of the most controversial issues in current bioethics and public health debates. For some, biobanks offer the possibility of unprecedented advances which will revolutionise research and improve the health of future generations. For others they are worrying repositories of personal information and tissue which will be used without sufficient respect for those from whom they came. Wherever one stands on this spectrum, from an ethics perspective biobanks are revolutionary. Traditional ethical safeguards of informed consent and confidentiality, for example, simply don’t work for the governance of biobanks and as a result new ethical structures are required. Thus it is not too great a claim to say that biobanks require a rethinking of our ethical assumptions and frameworks which we have applied generally to other issues in ethics. This paper maps the key challenges and controversies of biobanking ethics; it considers; informed consent (its problems in biobanking and possibilities of participants’ withdrawal), broad consent, the problems of confidentiality, ownership, property and comercialisation issues, feedback to participants and the ethics of re-contact

The Binary Protein Interactome of Treponema pallidum – The Syphilis Spirochete

Protein interaction networks shed light on the global organization of proteomes but can also place individual proteins into a functional context. If we know the function of bacterial proteins we will be able to understand how these species have adapted to diverse environments including many extreme habitats. Here we present the protein interaction network for the syphilis spirochete Treponema pallidum which encodes 1,039 proteins, 726 (or 70%) of which interact via 3,649 interactions as revealed by systematic yeast two-hybrid screens. A high-confidence subset of 991 interactions links 576 proteins. To derive further biological insights from our data, we constructed an integrated network of proteins involved in DNA metabolism. Combining our data with additional evidences, we provide improved annotations for at least 18 proteins (including TP0004, TP0050, and TP0183 which are suggested to be involved in DNA metabolism). We estimate that this “minimal” bacterium contains on the order of 3,000 protein interactions. Profiles of functional interconnections indicate that bacterial proteins interact more promiscuously than eukaryotic proteins, reflecting the non-compartmentalized structure of the bacterial cell. Using our high-confidence interactions, we also predict 417,329 homologous interactions (“interologs”) for 372 completely sequenced genomes and provide evidence that at least one third of them can be experimentally confirmed

DNA End Resection Controls the Balance between Homologous and Illegitimate Recombination in Escherichia coli

Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3′ ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD’s RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3′ tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3′ ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis

Effect of Ca2+ Channel Block on Glycerol Metabolism in Dunaliella salina under Hypoosmotic and Hyperosmotic Stresses

The effect of Ca2+ channel blockers on cytosolic Ca2+ levels and the role of Ca2+ in glycerol metabolism of Dunaliella salina under hypoosmotic or hyperosmotic stress were investigated using the confocal laser scanning microscope (CLSM). Results showed that intracellular Ca2+ concentration increased rapidly when extracellular salinity suddenly decreased or increased, but the increase could be inhibited by pretreatment of Ca2+ channel blockers LaCl3, verapamil or ruthenium red. The changes of glycerol content and G3pdh activity in D. salina to respect to hypoosmotic or hyperosmotic stress were also inhibited in different degrees by pretreatment of Ca2+ channel blockers, indicating that the influx of Ca2+ via Ca2+ channels are required for the transduction of osmotic signal to regulate osmotic responses of D. salina to the changes of salinity. Differences of the three blockers in block effect suggested that they may act on different channels or had different action sites, including influx of Ca2+ from the extracellular space via Ca2+ channels localized in the plasma membrane or from intracellular calcium store via the mitochondrial. Other Ca2+-mediated or non-Ca2+-mediated osmotic signal pathway may exist in Dunaliella in response to hypoosmotic and hyperosmotic stresses

Characterization of Multi-Functional Properties and Conformational Analysis of MutS2 from Thermotoga maritima MSB8

The MutS2 homologues have received attention because of their unusual activities that differ from those of MutS. In this work, we report on the functional characteristics and conformational diversities of Thermotoga maritima MutS2 (TmMutS2). Various biochemical features of the protein were demonstrated via diverse techniques such as scanning probe microscopy (SPM), ATPase assays, analytical ultracentrifugation, DNA binding assays, size chromatography, and limited proteolytic analysis. Dimeric TmMutS2 showed the temperature-dependent ATPase activity. The non-specific nicking endonuclease activities of TmMutS2 were inactivated in the presence of nonhydrolytic ATP (ADPnP) and enhanced by the addition of TmMutL. In addition, TmMutS2 suppressed the TmRecA-mediated DNA strand exchange reaction in a TmMutL-dependent manner. We also demonstrated that small-angle X-ray scattering (SAXS) analysis of dimeric TmMutS2 exhibited nucleotide- and DNA-dependent conformational transitions. Particularly, TmMutS2-ADPnP showed the most compressed form rather than apo-TmMutS2 and the TmMutS2-ADP complex, in accordance with the results of biochemical assays. In the case of the DNA-binding complexes, the stretched conformation appeared in the TmMutS2-four-way junction (FWJ)-DNA complex. Convergences of biochemical- and SAXS analysis provided abundant information for TmMutS2 and clarified ambiguous experimental results

Alcohol affects the emotional modulation of cognitive control: an event-related brain potential study

OBJECTIVE: The present study aimed to determine whether alcohol affects the emotional modulation of cognitive control and its underlying neural mechanisms, which is pivotal to an understanding of the socially maladaptive behaviors frequently seen in alcohol-intoxicated individuals. METHOD: Event-related potentials (ERPs) were recorded in male participants receiving either a moderate dose of alcohol (0.65 g/kg alcohol; n = 32) or a non-alcoholic placebo beverage (n = 32) while performing an emotional Go/No-Go task that required response execution (Go trials) to pictures of a “target” emotional facial expression (angry, happy, neutral) and response inhibition (No-Go trials) to a different “non-target” expression. RESULTS: Overall, N200 and P300 amplitudes were more enhanced during No-Go than Go trials. Interestingly, alcohol-intoxicated individuals displayed larger No-Go N200 amplitudes across all emotional conditions than controls, accompanied by decreased task performance (i.e., more errors), particularly in response to angry faces. P300 amplitude in the alcohol group was significantly reduced for both Go and No-Go trials, but only following angry and happy emotional expressions. CONCLUSIONS: These results suggest that alcohol-intoxicated individuals need to effortfully activate more cognitive resources during the early inhibition process in order to regulate a response than controls. Moreover, alcohol affected the emotional modulation of both response inhibition and execution in the later stages of cognitive control. Alcohol dampened emotional responsiveness, which may restrict the availability of attentional resources for cognitive control. Yet, these findings may underlie the lack of control in alcohol-intoxicated individuals when faced with emotionally or socially challenging situations