SUMMARY 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 per second. 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. Based upon these findings, we present a model describing the recruitment and regulation of Srs2 as it acts upon homologous recombination intermediates.
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.
We investigate the surface height fluctuations of single and double bilayers of DPPE supported on silicon using x-ray photon correlation spectroscopy (XPCS). In this techique, x-rays are incident on the membrane in a grazing incidence geometry and diffusely scattered x-rays are measured using an area detector. Time fluctuations of the scattering pattern can then be analyzed to yield the relaxation rate of surface height fluctuations. Bilayer and double bilayer systems were prepared utilizing combination of Langmuir-Blodgett and Langmuir-Schaeffer depositions. Static structural measurements were also made on these systems as well as on more complicated systems consisting of triple and five-fold bilayers of DPPE. Relationships between structure and dynamics of these systems will be discussed.
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 approximately 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 degrees 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.
Summary 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 nucleotides 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.
In recent years, new evidence in biomembrane research brought about a holistic, supramolecular view on membrane-mediated signal transduction. The consequences of sphingomyelinase (SMase)-driven formation of ceramide (Cer) at the membrane interface involves reorganization of the lateral membrane structure of lipids and proteins from the nm to the mum level. In this review, we present recent insights about mechanisms and features of the SMase-mediated formation of Cer-enriched domains in model membranes, which have been elucidated through a combination of microscopic techniques with advanced image processing algorithms. This approach extracts subtle morphological and pattern information beyond the visual perception: since domain patterns are the consequences of subjacent biophysical properties, a reliable quantitative description of the supramolecular structure of the membrane domains yields a direct readout of biophysical properties which are difficult to determine otherwise. Most of the information about SMase action on simple lipid interfaces has arisen from monolayer studies, but the correspondence to lipid bilayer systems will also be discussed. Furthermore, the structural changes induced by sphingomyelinase action are not fully explained just by the presence of ceramide but by out-of equilibrium surface dynamics forcing the lipid domains to adopt transient supramolecular pattern with explicit interaction potentials. This rearrangement responds to a few basic physical properties like lipid mixing/demixing kinetics, electrostatic repulsion and line tension. The possible implications of such transient codes for signal transduction are discussed for SMase controlled action on lipid interfaces.
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) SMenriched phase. Langmuir-Schaefer films were made in order to visualize the labeled enzyme. Independently of initial conditions Alexa-SMase is preferably localized in the SMenriched 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. Phospholipases are a group of mostly water-soluble enzymes, widely spread in nature, that perform their catalytic activity at an interface, owing to the insoluble nature of their substrates (1). Several studies have equated "membrane defects," such as those arising from the coexistence of lipid domains in different physical states, with enhanced catalytic activity (2-8). In 1990, it was reported that the liquidexpanded (LE), liquid-condensed (LC) lateral interfaces (lipid domain borders) in a one-component monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) act as starting points for Naja naja phospholipase A 2 (PLA 2 ) catalytic activity (9). Dahmen-Levison, Brezesinski, and Mohwald (10) showed in lipid monolayers that the fluorescent-labeled PLA 2 preferably accumulates at the LC-LE interface of domains. Those results support the socalled perimeter-activated or border-activated mechanism, in which the enzyme must have physical contact with the domain boundary/membrane defect in order to become fully active and exerts its major catalytic action adsorbed to these linear interfaces (11). However, more recent studies (12) have demonstrated that fluorescein-labeled PLA 2 from Crotalus atrox was homogeneously distributed in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and DPPC giant unilamellar vesicles when the lipids were in the LE state and preferentially localized in the LE phase at temperatures corresponding to the gel-fluid phase coexistence. The homogeneous distribution of PLA 2 on the membrane surface would support a mechanism by which the enzyme is fully active over the whole fluid surface (area-activated mechanism) but cannot adequately explain...
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.
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