dna complexes
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2022 ◽  
Petr Jurecka ◽  
Marie Zgarbova ◽  
Filip Cerny ◽  
Jan Salomon

When DNA interacts with a protein, its structure often undergoes significant conformational adaptation. Perhaps the most common is the transition from canonical B-DNA towards the A-DNA form, which is not a two-state, but rather a continuous transition. The A- and B- forms differ mainly in sugar pucker P (north/south) and glycosidic torsion χ (high-anti/anti). The combination of A-like P and B-like χ (and vice versa) represents the nature of the intermediate states lying between the pure A- and B- forms. In this work, we study how the A/B equilibrium and in particular the A/B intermediate states, which are known to be over-represented at protein-DNA interfaces, are modeled by current AMBER force fields. Eight protein-DNA complexes and their naked (unbound) DNAs were simulated with OL15 and bsc1 force fields as well as an experimental combination OL15χOL3. We found that while the geometries of the A-like intermediate states in the molecular dynamics (MD) simulations agree well with the native X-ray geometries found in the protein-DNA complexes, their populations (stabilities) are significantly underestimated. Different force fields predict different propensities for A-like states growing in the order OL15 < bsc1 < OL15χOL3, but the overall populations of the A-like form are too low in all of them. Interestingly, the force fields seem to predict the correct sequence-dependent A-form propensity, as they predict larger populations of the A-like form in naked (unbound) DNA in those steps that acquire A-like conformations in protein-DNA complexes. The instability of A-like geometries in current force fields may significantly alter the geometry of the simulated protein-DNA complex, destabilize the binding motif, and reduce the binding energy, suggesting that refinement is needed to improve description of protein-DNA interactions in AMBER force fields.

2021 ◽  
Vol 23 (1) ◽  
pp. 212
Sridhar Vemulapalli ◽  
Mohtadin Hashemi ◽  
Yuri L. Lyubchenko

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM (HS-AFM) to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualised sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.

2021 ◽  
pp. 106950
Beth A. Bouchard ◽  
Christos Colovos ◽  
Michael A. Lawson ◽  
Zachary T. Osborn ◽  
Adrian M. Sackheim ◽  

2021 ◽  
pp. 1-10
Meera Thomas ◽  
Anindya Chowdhury ◽  
Amit K. Majhi ◽  
V. A. Raghunathan

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 437-437
Frederik Denorme ◽  
Irina Portier ◽  
Mark Cody ◽  
Ramesh Grandhi ◽  
Matthew D Neal ◽  

Abstract Background: Ischemic stroke provokes a strong inflammatory response which has been shown to exacerbate ischemic stroke brain injury in mice and is associated with worse outcomes in ischemic stroke patients. Classic anti-inflammatory strategies have been unsuccessful in clinical trials for ischemic stroke, implying other mechanisms contribute to injurious inflammation in ischemic stroke. Immunothrombosis is the interplay between coagulation, platelet activation and the innate immune system leading to thrombosis. A critical component of immunothrombosis is the formation of neutrophil extracellular traps (NETs). In this study, we investigated mechanistic regulators of NET formation in stroke and if they contribute to ischemic stroke outcomes. Methods: Markers of immunothrombosis were assessed in plasma from ischemic stroke patients and healthy matched donors. Flow cytometry was used to characterize platelet and neutrophil function. For murine studies, we used male and female C57Bl/6 mice that were subjected to transient middle cerebral artery occlusion. Stroke outcomes were assessed 24 hours or 7 days after stroke using neurological and motor function testing as well as brain infarct size analysis. Results: Ischemic stroke patients had significantly increased plasma biomarkers of immunothrombosis including D-Dimers (coagulation), platelet factor 4 (platelet activation) and calprotectin (neutrophil activation). Moreover, specific markers of NET formation including citrullinated histone H3 (H3cit) and MPO-DNA complexes were significantly elevated in ischemic stroke patients. Interestingly, H3cit and MPO-DNA complexes positively correlated with worse stroke outcomes at discharge while they did not correlate with stroke severity at admission. Next, we observed increased plasma and platelet surface expressed high mobility group box 1 (HMGB1) in ischemic stroke patients compared to matched healthy controls. NETs were found in platelet-rich areas in ischemic stroke thrombi, and HMGB1 colocalized with platelets in ischemic stroke thrombi. Blocking platelet-derived HMGB1 in vitro prevented platelet-induced NET formation. Mechanistically, depleting platelets in mice reduced plasma HMGB1 levels as well as NET formation and improved outcomes after ischemic stroke. In contrast, depleting neutrophils did not affect plasma HMGB1 levels, but reduced plasma NETs and improved stroke outcomes. Treatment of mice with a competitive HMGB1 inhibitor (BoxA) reduced NET formation and improved stroke outcomes. Combined, these results imply a causative role for platelet-derived HMGB1 in mediating detrimental NET formation after ischemic stroke. Finally, as NETs appeared injurious in ischemic stroke, we investigated the therapeutic potential of a recently discovered neonatal NET inhibitory factor (nNIF). nNIF is a cleavage fragment of alpha-1-antitrypsin and specifically blocks NET formation in human and murine neutrophils without affecting other critical neutrophil functions such as chemotaxis or phagocytosis. Mice treated with nNIF had reduced brain and plasma NETs after stroke while cerebral neutrophil recruitment remained unaffected. The reduction in NET formation after stroke was associated with reduced neuronal apoptosis and smaller brain infarcts. Furthermore, nNIF treated mice had improved neurological and motor function and enhanced 7-day survival after ischemic stroke. Conclusions: These results support a pathological role for NETs in acute ischemic stroke and warrant further investigation into nNIF as a therapeutic to improve stroke outcomes. Disclosures Neal: Instrumentation Laboratories: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Haima Therapeutics: Membership on an entity's Board of Directors or advisory committees; Haemonetics: Honoraria, Research Funding.

Mark Underwood ◽  
Joe Horton ◽  
Keith Nangle ◽  
Judy Hopking ◽  
Kimberly Smith ◽  

At Week 48 in the phase IIIb DAWNING study, the integrase strand transfer inhibitor (INSTI) dolutegravir plus 2 nucleoside reverse transcriptase inhibitors demonstrated superiority to ritonavir-boosted lopinavir in achieving virologic suppression in adults with HIV-1 who failed first-line therapy. Here we report emergent HIV-1 drug resistance and mechanistic underpinnings among dolutegravir-treated adults in DAWNING. Population viral genotyping, phenotyping, and clonal analyses were performed on participants meeting confirmed virologic withdrawal (CVW) criteria on dolutegravir-containing regimens. Dolutegravir binding to and structural changes in HIV-1 integrase-DNA complexes with INSTI resistance-associated substitutions were evaluated. Of participants who received dolutegravir through Week 48 plus an additional 110 weeks for this assessment, 6 met CVW criteria with treatment-emergent INSTI resistance-associated substitutions and 1 had R263R/K at baseline but not at CVW. All 7 achieved HIV-1 RNA <400 copies/mL (5 achieved <50 copies/mL) before CVW. Treatment-emergent G118R was detected in 5 participants, occurring with ≥2 other integrase substitutions, including R263R/K, in 3 participants and without other integrase substitutions in 2 participants. G118R or R263K increased the dolutegravir dissociation rate from integrase-DNA complexes vs wild-type but retained prolonged binding. Overall, among treatment-experienced adults who received dolutegravir in DAWNING, 6 of 314 participants developed treatment-emergent INSTI resistance-associated substitutions, with in vitro dolutegravir resistance >10 fold-change and reduced viral replication capacity vs baseline levels. This study demonstrates that the pathway to dolutegravir resistance is a challenging balance between HIV-1 phenotypic change and associated loss of viral fitness (ClinicalTrials.gov identifier: NCT02227238).

2021 ◽  
Vol 22 (21) ◽  
pp. 11323
Carolina Gándara ◽  
Rubén Torres ◽  
Begoña Carrasco ◽  
Silvia Ayora ◽  
Juan C. Alonso

DNA lesions that impede fork progression cause replisome stalling and threaten genome stability. Bacillus subtilis RecA, at a lesion-containing gap, interacts with and facilitates DisA pausing at these branched intermediates. Paused DisA suppresses its synthesis of the essential c-di-AMP messenger. The RuvAB-RecU resolvasome branch migrates and resolves formed Holliday junctions (HJ). We show that DisA prevents DNA degradation. DisA, which interacts with RuvB, binds branched structures, and reduces the RuvAB DNA-dependent ATPase activity. DisA pre-bound to HJ DNA limits RuvAB and RecU activities, but such inhibition does not occur if the RuvAB- or RecU-HJ DNA complexes are pre-formed. RuvAB or RecU pre-bound to HJ DNA strongly inhibits DisA-mediated synthesis of c-di-AMP, and indirectly blocks cell proliferation. We propose that DisA limits RuvAB-mediated fork remodeling and RecU-mediated HJ cleavage to provide time for damage removal and replication restart in order to preserve genome integrity.

Polymer ◽  
2021 ◽  
pp. 124277
Aleksandra Radko ◽  
Sebastian Lalik ◽  
Aleksandra Deptuch ◽  
Teresa Jaworska-Gołąb ◽  
Robert Ekiert ◽  

2021 ◽  
pp. 117930
Filipe Coelho ◽  
Claudia Botelho ◽  
Juan L. Paris ◽  
Eduardo F. Marques ◽  
Bruno F. B. Silva

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2545
Jiram Torres-Ruiz ◽  
Abdiel Absalón-Aguilar ◽  
Miroslava Nuñez-Aguirre ◽  
Alfredo Pérez-Fragoso ◽  
Daniel Alberto Carrillo-Vázquez ◽  

The coronavirus disease 2019 (COVID-19) is related to enhanced production of NETs, and autoimmune/autoinflammatory phenomena. We evaluated the proportion of low-density granulocytes (LDG) by flow cytometry, and their capacity to produce NETs was compared with that of conventional neutrophils. NETs and their protein cargo were quantified by confocal microscopy and ELISA. Antinuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and the degradation capacity of NETs were addressed in serum. MILLIPLEX assay was used to assess the cytokine levels in macrophages’ supernatant and serum. We found a higher proportion of LDG in severe and critical COVID-19 which correlated with severity and inflammatory markers. Severe/critical COVID-19 patients had higher plasmatic NE, LL-37 and HMGB1-DNA complexes, whilst ISG-15-DNA complexes were lower in severe patients. Sera from severe/critical COVID-19 patients had lower degradation capacity of NETs, which was reverted after adding hrDNase. Anti-NET antibodies were found in COVID-19, which correlated with ANA and ANCA positivity. NET stimuli enhanced the secretion of cytokines in macrophages. This study unveils the role of COVID-19 NETs as inducers of pro-inflammatory and autoimmune responses. The deficient degradation capacity of NETs may contribute to the accumulation of these structures and anti-NET antibodies are related to the presence of autoantibodies.

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