Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous ...recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.
Srs2 is a superfamily 1 (SF1) helicase and antirecombinase that is required for genome integrity. However, the mechanisms that regulate Srs2 remain poorly understood. Here, we visualize Srs2 as it ...acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. We demonstrate that Srs2 is a processive translocase capable of stripping thousands of Rad51 molecules from ssDNA at a rate of ∼50 monomers/s. We show that Srs2 is recruited to RPA clusters embedded between Rad51 filaments and that multimeric arrays of Srs2 assemble during translocation on ssDNA through a mechanism involving iterative Srs2 loading events at sites cleared of Rad51. We also demonstrate that Srs2 acts on heteroduplex DNA joints through two alternative pathways, both of which result in rapid disruption of the heteroduplex intermediate. On the basis of these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.
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•Srs2 is visualized in real time as it translocates on Rad51-ssDNA•Srs2 rapidly strips Rad51 from ssDNA•Srs2 loads preferentially at RPA clusters embedded between Rad51 filaments•Srs2 rapidly disrupts small heteroduplex DNA joints bound to Rad51 filaments
Kaniecki et al. develop a single-molecule assay for directly visualizing the behavior of the yeast helicase Srs2 as it acts upon single-stranded DNA (ssDNA) bound by the Rad51 recombinase. These experiments lead to a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.
Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves ...recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.
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•Srs2 is visualized in real time as it translocates on ssDNA•Srs2 is able to translocate rapidly on ssDNA coated with RPA•Srs2 can also translocate on ssDNA bound by both RPA and Rad52•RPA and Rad52 are removed from ssDNA by Srs2
De Tullio et al. develop a single-molecule assay for directly visualizing the behavior of the yeast helicase Srs2 as it acts upon single-stranded DNA (ssDNA). These experiments demonstrate that Srs2 is capable of rapid and processive translocation on ssDNA coated with the homologous recombination accessory proteins RPA and Rad52.
The hallmark of the mismatch repair system in bacterial and eukaryotic organisms devoid of MutH is the presence of a MutL homologue with endonuclease activity. The aim of this study was to analyse ...whether different DNA structures affect Pseudomonas aeruginosa MutL (PaMutL) endonuclease activity and to determine if a specific nucleotide sequence is required for this activity. Our results showed that PaMutL was able to nick covalently closed circular plasmids but not linear DNA at high ionic strengths, while the activity on linear DNA was only found below 60 mM salt. In addition, single strand DNA, ss/ds DNA boundaries and negatively supercoiling degree were not required for PaMutL nicking activity. Finally, the analysis of the incision sites revealed that PaMutL, as well as Bacillus thuringiensis MutL homologue, did not show DNA sequence specificity.
A combination of lipid monolayer- and bilayer-based model systems has been applied to explore in detail the interactions between and organization of palmitoylsphingomyelin (pSM) and the related lipid ...palmitoylceramide (pCer). Langmuir balance measurements of the binary mixture reveal favorable interactions between the lipid molecules. A thermodynamically stable point is observed in the range ∼30–40 mol % pCer. The pSM monolayer undergoes hyperpolarization and condensation with small concentrations of pCer, narrowing the liquid-expanded (LE) to liquid-condensed (LC) pSM main phase transition by inducing intermolecular interactions and chain ordering. Beyond this point, the phase diagram no longer reveals the presence of the pSM-enriched phase. Differential scanning calorimetry (DSC) of multilamellar vesicles reveals a widening of the pSM main gel-fluid phase transition (41°C) upon pCer incorporation, with formation of a further endotherm at higher temperatures that can be deconvoluted into two components. DSC data reflect the presence of pCer-enriched domains coexisting, in different proportions, with a pSM-enriched phase. The pSM-enriched phase is no longer detected in DSC thermograms containing >30 mol % pCer. Direct domain visualization has been carried out by fluorescence techniques on both lipid model systems. Epifluorescence microscopy of mixed monolayers at low pCer content shows concentration-dependent, morphologically different pCer-enriched LC domain formation over a pSM-enriched LE phase, in which pCer content close to 5 and 30 mol % can be determined for the LE and LC phases, respectively. In addition, fluorescence confocal microscopy of giant vesicles further confirms the formation of segregated pCer-enriched lipid domains. Vesicles cannot form at >40 mol % pCer content. Altogether, the presence of at least two immiscible phase-segregated pSM-pCer mixtures of different compositions is proposed at high pSM content. A condensed phase (with domains segregated from the liquid-expanded phase) showing enhanced thermodynamic stability occurs near a compositional ratio of 2:1 (pSM/pCer). These observations become significant on the basis of the ceramide-induced microdomain aggregation and platform formation upon sphingomyelinase enzymatic activity on cellular membranes.
We describe the localization of Alexa-488-labeled SMase in SM/ceramide (Cer) lipid monolayers containing segregated liquid-condensed (LC) Cer-enriched domains surrounded by a continuous ...liquid-expanded (LE) SM-enriched phase. Langmuir-Schaefer films were made in order to visualize the labeled enzyme. Independently of initial conditions Alexa-SMase is preferably localized in the SM-enriched LE phase and it is not enriched at the domain boundaries. A novel mechanism is proposed for the action of SMase, which can also explain the regulatory effect of the surface topography on the enzyme activity. The homogeneous enzymatic generation of Cer in the LE phase leads to a meta-stable, kinetically trapped, supersaturated mixed monolayer. This effect acts as driving force for the segregation of the Cer-enriched domain following classical nucleation mechanisms. Accordingly, the number and size of Cer-enriched domains are determined by the extent of Cer supersaturation in the LE phase rather than by the SMase local activity. The kinetic barrier for nucleation, for which a compositional gap of at least 53 mol% of Cer is necessary to reach a thermodynamically stable LC phase, can explain the lag time to reaching full catalytic activity. Altogether, the data support an “area-activated mechanism,” in which the enzyme is homogeneously active over the LE surface.
Helicases are crucial participants in many types of DNA repair reactions, including homologous recombination. The properties of these enzymes can be assayed by traditional bulk biochemical analysis; ...however, these types of assays cannot directly access some types of information. In particular, bulk biochemical assays cannot readily access information that may be obscured in population averages. Single-molecule assays offer the potential advantage of being able to visualize the molecules in question in real time, thus providing direct access to questions relating to translocation velocity, processivity, and insights into how helicases may behave on different types of substrates. Here, we describe the use of single-stranded DNA (ssDNA) curtains as an assay for directly viewing the behavior of the Saccharomyces cerevisiae Srs2 helicase on single molecules of ssDNA. When used with total internal reflection fluorescence microscopy, these methods can be used to track the binding and movements of individual helicase complexes, and allow new insights into helicase behaviors at the single-molecule level.
Biomembranes contain a wide variety of lipids and proteins within an essentially two-dimensional structure. The coexistence of such a large number of molecular species causes local tensions that ...frequently relax into a phase or compositional immiscibility along the lateral and transverse planes of the interface. As a consequence, a substantial microheterogeneity of the surface topography develops and that depends not only on the lipid–protein composition, but also on the lateral and transverse tensions generated as a consequence of molecular interactions. The presence of proteins, and immiscibility among lipids, constitute major perturbing factors for the membrane sculpturing both in terms of its surface topography and dynamics. In this work, we will summarize some recent evidences for the involvement of membrane-associated, both extrinsic and amphitropic, proteins as well as membrane-active phosphohydrolytic enzymes and sphingolipids in driving lateral segregation of phase domains thus determining long-range surface topography.
Changes of the initial composition and topography of mixed monolayers of Sphingomyelin and Ceramide modulate the degradation of Sphingomyelin by Bacillus cereus Sphingomyelinase. The presence of ...initial lateral phase boundary due to coexisting condensed and expanded phase domains favors the precatalytic steps of the reaction. The amount and quality of the domain lateral interface, defined by the type of boundary undulation, appears as a modulatory supramolecular code which regulates the catalytic efficiency of the enzyme. The long range domain lattice structuring is determined by the Sphingomyelinase activity.
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Background: In non-small cell lung cancer (NSCLC), the MET tyrosine kinase receptor can be dysregulated by mutations and/or gene amplification. The most common MET mutation is in exon 14 ...(METex14), leading to impaired receptor degradation and increased MET-mediated signaling causing sustained tumor proliferation. MET amplification also leads to continued MET signaling and oncogenesis and can be a mechanism of resistance to targeted therapy. We sought to compare the genomic landscape of METex14 and high-level MET amplified tumors, both of which can be targeted with tyrosine kinase inhibitors. Methods: We analyzed 18,047 NSCLC tumors (any stage/subtype) sequenced with Tempus xT assay (DNA-seq of 648 genes at 500x coverage, full transcriptome RNA-seq). Tumors were queried for METex14 mutations, high MET amplification (METamp), defined as copy number gain (CNG) ≥10, and other MET mutations (METother). Immuno-oncology (IO) biomarkers were compared across MET-altered and MET wild-type (METwt) groups. The prevalence of somatic gene alterations was compared similarly with the false-discovery rate (FDR) adjustment. Results: A total of 276 (1.53%) METex14, 138 (0.76%) METamp, 27 (0.15%) METother, and 17,606 (97.56%) METwt tumors were identified. Across groups, patients with METex14 were older, more likely to be female, and nonsmokers. METex14 exhibited the lowest tumor mutational burden (TMB) and lowest neoantigen tumor burden (NTB). PD-L1 positivity rates were higher in METex14 tumors compared to METamp and METwt tumors. MET log10 gene expression was highest in METamp. METamp exhibited the lowest proportion of CD4 T cells and the highest proportion of NK cells. Compared to METex14, METamp exhibited increased prevalence of TP53 (83% vs 37%), TFEC (46% vs 1.1%), CFTR (37% vs 1.4%), EGFR (31% vs. 7.6%), WNT2 (11% vs 0.4%), KEAP1 (14% vs 0.7%), and STK11 (8% vs 1.4%), and decreased prevalence of MDM2 (4.3% vs. 14%), and FRS2 (2.9% vs. 12%). Conclusions: In this large population-based analysis of MET-altered NSCLC, METex14 tumors exhibited differences in IO biomarkers and the somatic landscape compared to non-METex14 NSCLC tumors. Variations in immune profiles may affect immunotherapy selection in MET-altered NSCLC and require further exploration. Table: see text
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