Investigation of the DNA binding domain of nuclear factor I

Nuclear Factor I is a cellular transcription factor involved in the initiation of adenovirus type 2 replication. It exists as a family of proteins and comprises two distinct domains. The N-terminal domain is highly conserved between species and is involved in DNA binding, dimerisation, and stimulates adenovirus replication. The C terminal domain is responsible for the transcriptional activation. In this work, the N terminal, DNA binding domain of Nuclear Factor I, was cloned in two different expression systems. Using recombinant baculovirus, the unmodified protein and a GST fusion product were expressed in insect cells. Although both proteins were expressed at high levels, it was impossible to purify the fusion protein, while after purification, the unmodified protein remained heterogeneous. Expression in E. coli using the expression vector pET22b and pGEX2T produced inclusion bodies. The insoluble material was extracted, solubilised using guanidine HCl, then folded in vitro using new additives known as non-detergent sulphobetaines. In vitro folding was optimised and yields of up to 8% could be obtained. The affinity of the refolded material for a specific DNA oligonucleotide was determined in a gel electrophoresis DNA binding assay and was identical to the native protein purified from baculovirus infected insect cells. The structure of the Nuclear Factor I DNA Binding Domain was investigated using limited proteolysis followed by separation on SDS PAGE, N terminal sequencing and mass spectrometry. Residues 1 to 165 formed a compact domain. Residues 166 - 181 were extremely sensitive to degradation in the absence of DNA but fully protected in the presence of specific DNA. The C terminal region from residue 182 could be degraded in both conditions. Along with two other regions previously determined which bind DNA, the region between residues 166 and 181 is required for DNA binding. Differential labelling using iodoacetate showed that cysteine residues 95 and 111 were modified in the free protein which resulted in an inactive protein but were protected in the presence of DNA. This demonstrates their direct involvement in DNA binding

Human ANKLE1 is a nuclease specific for branched DNA

All physical connections between sister chromatids must be broken before cells can divide, and eukaryotic cells have evolved multiple ways in which to process branchpoints connecting DNA molecules separated both spatially and temporally. A single DNA link between chromatids has the potential to disrupt cell cycle progression and genome integrity, so it is highly likely that cells require a nuclease that can process remaining unresolved and hemi-resolved DNA junctions and other branched species at the very late stages of mitosis. We argue that ANKLE1 probably serves this function in human cells (LEM-3 in Caenorhabditis elegans). LEM-3 has previously been shown to be located at the cell mid-body, and we show here that human ANKLE1 is a nuclease that cleaves a range of branched DNA species. It thus has the substrate selectivity consistent with an enzyme required to process a variety of unresolved and hemi-resolved branchpoints in DNA. Our results suggest that ANKLE1 acts as a catch-all enzyme of last resort that allows faithful chromosome segregation and cell division to occur. (C) 2020 The Author(s). Published by Elsevier Ltd

Beyond the Comfort Zone: A Guide to Supervising Qualitative Undergraduate Psychology Dissertations for Quantitative Researchers

The Teaching Qualitative Psychology Group (TQP) is a group of experienced academics supporting the sharing of best practice in the teaching and supervision of qualitative research methods in psychology. In this paper the group share their knowledge and practice suggestions with a specific focus on supporting academics who do not come from a qualitative research background, but who are supervising qualitative dissertations. This paper will explore why quantitative researchers may want to supervise qualitative dissertations and suggest ways in which this methodological shift might be managed well in the context of the undergraduate project as well as some practical advice for a valuable supervision experience

Does treating insomnia with digital cognitive behavioural therapy (Sleepio) mediate improvements in anxiety for those with insomnia and comorbid anxiety?:An analysis using individual participant data from two large randomised controlled trials

Background: Considerable comorbidity exists between insomnia and anxiety, and evidence shows that the benefits of CBT for insomnia extend to anxiety. Using data from two large trials of digital CBT (dCBT) for insomnia, we evaluated whether improving sleep is an effective treatment target to reduce both insomnia and anxiety symptoms in individuals with insomnia and clinically significant anxiety. Methods: This was a controlled sub-analysis combining individual participant data from two previous randomised controlled trials of dCBT for insomnia (Sleepio). Participants (N = 2172) with insomnia disorder and clinically significant anxiety symptoms were included in this sub-analysis and received either dCBT or control (usual care or sleep hygiene education). Assessments were evaluated at baseline, post-intervention (week 8 or 10), and follow-up (week 22 or 24). Mediation was evaluated using structural equation models. Results: dCBT for insomnia was superior to control at reducing both insomnia (Hedges' g range = 0.77–0.81; both p &lt; 0.001) and anxiety symptoms (Hedges' g range = 0.39–0.44; both p &lt; 0.001) at all time points. Baseline insomnia symptoms moderated the effects of dCBT on insomnia, however no variables moderated treatment effects on anxiety. Reductions in anxiety symptoms at follow-up were mediated by improvements in sleep at post-intervention (% mediated = 84 %), suggesting a causal pathway. Limitations: Participants did not have a formal anxiety disorder diagnosis and so the effects of dCBT for insomnia on anxiety may differ by anxiety disorder. Conclusions: Addressing sleep using dCBT for insomnia may serve as a treatment target from which to improve anxiety in individuals with insomnia and clinically significant comorbid anxiety. Clinical trial registrations: Digital Insomnia therapy to Assist your Life as well as your Sleep (DIALS) - ISRCTN60530898 http://www.isrctn.com/ISRCTN60530898. Oxford Access for Students Improving Sleep (OASIS) - ISRCTN61272251 http://www.isrctn.com/ISRCTN61272251.</p

Junction resolving enzymes use multivalency to keep the Holliday junction dynamic

Holliday junction (HJ) resolution by resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single-molecule fluorescence resonance energy transfer analysis of resolving enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.11Nsciescopu

GEN1 from a thermophilic fungus is functionally closely similar to non-eukaryotic junction-resolving enzymes

AbstractProcessing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3′ to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1

Analysis of the Intrinsically Disordered N-Terminus of the DNA Junction-Resolving Enzyme T7 Endonuclease I:Identification of Structure Formed upon DNA Binding

This work was supported by grants from The Engineering and Physical Sciences Research Council (EPSRC), Basic Technology EP/F039034/1, The Wellcome Trust, 099149/Z/12/Z, and Cancer Research UK (CRUK), C28/A18604.The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7-10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2-12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography.Publisher PDFPeer reviewe

The effects of acute interval exercise and strawberry intake on postprandial lipemia

YesPurpose: Raised postprandial triglycerides (TAG) and related oxidative stresses are strongly associated with increased cardiovascular disease (CVD) risk. Acute exercise and strawberry ingestion independently ameliorate postprandial lipid excursions and oxidative stress. However, the combined effects of these lifestyle interventions is unknown. We investigated whether acute exercise and strawberry consumption improved postprandial responses to an oral fat tolerance test (OFTT) in overweight/obese males. Methods: Overweight/obese adult males underwent four separate OFTT (73g fat, 33g carbohydrate) with blood sampled at baseline and hourly for 4 h after OFTT. Two OFTT contained 25g freeze-dried strawberries and two contained strawberry flavouring (placebo). Participants performed 40 minutes of submaximal high intensity interval cycling exercise (HIIE) 16 h before one strawberry and one placebo OFTT, and rested before the remaining two OFTT. Serum TAG was analysed and TAG area under curve (AUC) and incremental AUC (iAUC) were calculated. Oxidative stress markers were measured at baseline and 4 h. Differences between conditions (strawberry/placebo and exercise/rest) were assessed using repeated measures ANOVA. Results: Ten males (Age, 31.5 IQR 17.8 years; BMI, 29.9 ±1.8 kg.m-2) completed the study. TAG AUC was 1.5 mmol.4h-1.L-1 lower for the exercise conditions compared to the rest conditions (95% confidence interval [CI]= -2.3 to 0.8, p= 0.001). TAG AUC was not different between the strawberry and placebo conditions (CI= -1.3 to 0.6, p= 0.475). TAG iAUC was 0.5 mmol.4h-1.L-1 greater for the strawberry compared to the placebo conditions (CI= 0.1 to 1.0, p= 0.021). There were no changes in markers of lipid related oxidative stress (P> 0.05). Conclusion: Acute submaximal HIIE appears effective in reducing postprandial lipaemia in overweight/obese adult males. However, strawberry ingestion did not improve postprandial TAG