Cells must protect their genome from harmful alterations, caused directly by DNA damage or indirectly by inappropriate repair, to avoid cell death or cancerous transformation. DNA double-strand (ds) breaks, the most severe type of DNA damage, are repaired in a potentially error-free manner by homologous recombination (HR). During HR, broken dsDNA ends are transformed into 3' single-stranded (ss) overhangs that form nucleoprotein filaments with recombinases. These filaments can search for intact, homologous dsDNA regions that serve as template for repair and can invade into the homologous DNA molecule to form a displacement loop (D-loop). D-loops are thus key HR DNAintermediate structures. D-loops are thought to be processed by multiple pathways with different outcomes and can serve as early decision points in HR regulation. Members of the RecQ helicase family are implicated both in D-loop stabilization and disruption. The human Bloom's syndrome RecQ helicase (BLM) is thought to channel HR into error-free pathways partially by processing D-loops. However, the exact mechanism of this action is poorly understood. Via a novel kinetic assay, here we show that the conserved domain architecture of BLM maintains an almost 1:1 balance in D-loop disruption and stabilization. Interestingly, the activity profile of BLM markedly differs from that of the very effective D-loop disruptor Escherichia coli RecQ. However, our single-molecule magnetic tweezers experiments reveal that the similar domain architecture of BLM and RecQ, while having different roles in D-loop processing, allows both proteins to sense DNA substrate geometry and unwind dsDNA in a repetitive fashion. Our results highlight how functions of the conserved architecture of RecQ helicases evolved to specialize these enzymes to different cellular requirements.
Objective Oxysterols such as 25‐hydroxycholesterol (25HC) are metabolites of cholesterol produced in the human body. Oxysterols are implicated in many physiological and pathological processes and are shown to regulate the immune system. Recently, we demonstrated that during virus infections, 25HC amplifies proinflammatory response by directly binding to αvβ3 and α5β1 integrins and by activating FAK‐NFkB signaling pathway, resulting in the production of proinflammatory mediators such as TNFα and IL‐6. Surprisingly, our combined in silico and in vitro studies revealed that 25HC binds to integrins at a novel binding site (site 2) that is distinct from the classical ‘RGD’ binding site, where many extracellular matrix proteins with Arg‐Gly‐Asp motif are known to bind. Integrins are ubiquitous and play an important role in cell adhesion, cell‐cell interactions, cell signaling, and extracellular matrix assembly. Given the abundance of both integrins and endogenous oxysterols, we hypothesized that other oxysterols (other than 25HC) might also activate integrins in a similar fashion. In this study, using a combined computational and experimental approach, we show that 24(S)‐hydroxycholesterol (24HC) binds to integrin αvβ3 with high‐affinity and triggers proinflammatory response via integrin‐FAK pathway. Methods Molecular docking and all‐atom molecular dynamics (MD) simulations were used to obtain binding poses and to study the ligand stability and conformational changes, respectively. Induced fit docking of 24HC at site 2 of αVβ3 ectodomain (PDB ID 3IIJE) was performed using MOE software. All‐atom MD simulations were performed with GROMACS v5.1.2 using CHARMM36 force field and TIP3P water model. MD simulations were run in triplicates each for 200 ns. The proinflammatory response was determined by the production of TNF using ELISA. Results Our studies reveal the preferred binding orientation of 24HC, which is distinct from that of 25HC. Binding of 24HC induced structural changes in the specificity determining loop (SDL) of the βI domain, leading to an increase in solvent‐accessible surface area (SASA) of the RGD site, disruption of native SDL‐βI and SDL‐β‐propeller interface contacts, possibly altering the conformation of the RGD binding site. Robust production of TNF analyzed by ELISA indicates activation of the inflammatory response by 24HC. Similar to 25HC, 24HC also requires integrin‐FAK pathway for inflammatory response since, FAK inhibitor, PF‐431396 abrogated 24HC mediated response. Conclusions: As the primary cholesterol metabolite with exclusive production in the brain, these findings may provide key insight into factors that influence the complexities of brain function. Our study, for the first time, offers useful insight into the structural and mechanistic bases for the potential proinflammatory response mediated by 24HC through integrins and paves the way for drug discovery efforts towards identifying therapeutics in the treatment of infectious, inflammatory, and immune disorders.
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