A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

30 pags., 10 figs., 1 tab.Telomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.Cancer Council NSW RG 11-07 Tracy M Bryan, Cancer Institute NSW Aaron Lavel Moye, Australian Research Council FL140100027 Antoine M van Oijen, Ernest and Piroska Major Foundation Scott B Cohen, Natural Sciences and Engineering Research Council of Canada, Masad J Damha Centre of Excellence for Innovation in Chemistry PERCH-CIC Siritron Samosorn Research Unit of Natural Products and Organic Synthesis for Drug Discovery NPOS 405/2560 Siritron Samosorn Cancer Council NSW RG 16-10 Tracy M Brya

i-Motif DNA: Structural Features and Significance to Cell Biology

19 pags., 7 figs.The i-motif represents a paradigmatic example of the wide structural versatility of nucleic acids. In remarkable contrast to duplex DNA, i-motifs are four-stranded DNA structures held together by hemi- protonated and intercalated cytosine base pairs (C:C+). First observed 25 years ago, and considered by many as a mere structural oddity, interest in and discussion on the biological role of i-motifs have grown dramatically in recent years. In this review we focus on structural aspects of i-motif formation, the factors leading to its stabilization and recent studies describing the possible role of i-motifs in fundamental biological processes.Natural Sciences and Engineering Research Council of Canada (NSERCC); MINECO grant [BFU2017-89707-P]; Juan de la Cierva postdoctoral Fellowship [FJCI-2016-28474 to M.G.]. Funding for open access charge: NSERCC Discovery grant ( to M.J.D.); MINECO grant [BFU2017-89707-P to C.G.].Peer Reviewe

2'-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2'-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.Peer Reviewe

Damha. Mapping the Affinity Landscape of Thrombin-binding Aptamers on 2'F-ANA/DNA Chimeric G-Quadruplex Microarrays

Swiss National Science Foundation [PBBEP2-146174 to J.L.]; Natural Sciences and Engineering Research Council of Canada [to M.J.D.]; Austrian Science Fund [FWF P23797 and FWF P27275 to J.L. and M.M.S.]; Spanish MINECO [BFU2014-52864-R to C.G.]. Funding for open access charge: Austrian Science Fund [FWF P23797 and FWF P27275 to J.L. and M.M.S.].Peer Reviewe

Conservative approach in the management of severe acute pancreatitis: eight-year experience in a single institution

Introduction. Recognition of severe acute pancreatitis (SAP), intensive care, shifting away from early surgical treatment, and monitoring of the intra-abdominal pressure (IAP) is important in the management of SAP. The aim of our study was retrospective evaluation and critical assessment of the experience with SAP management protocol involving new strategy in the university hospital. Methods. Protocols of 274 SAP patients treated in our institution during the last eight years were reassessed. APACHE II, CRP and SOFA score, IAP, pulmonary complications, ventilatory support and infection rate were evaluated. The success of the conservative treatment, surgical interventions and mortality was analysed comparing period 1 from 1999 to 2002 and period 2 from 2003 to 2006. Results. More patients with necrotising SAP were treated in period 2. The average CRP and SOFA score was higher in period 2, p=0.018; p=0.011. A total of 139 patients underwent continuous veno-venouse haemofiltration (CVVH) as a component of fluid resuscitation and IAP control. Application of CVVH increased in period 2, p<0.005. Only 5–8% of patients were managed with ventilatory support. The overall infection rate decreased in period 2 comprising 21%, p<0.005. Success rate of the conservative therapy reached 69% in period 2, p<0.01. Surgical treatment was performed in 41% of patients in period 1 vs. 19% in period 2, p<0.001. Overall mortality was 19%, with a reduction to 12% in year 2006. Conclusion. The conservative protocol-based approach is a rational treatment strategy for the management of SAP and can be successfully implemented in the setting of the university hospital