Combining atomic force and fluorescence microscopy for analysis of quantum‐dot labeled protein–DNA complexes

Ebenstein, Yuval; Gassman, Natalie R.; Kim, Soo-Hong; Weiss, Shimon

doi:10.1002/jmr.956

Cited by 26 publications

(29 citation statements)

References 24 publications

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“…First, flatness and transparency of the sample, which we achieved by gluing a few layers of mica to a thin glass coverslip (6,20). The advantages over using only glass (4,30) or only mica (5) are the improved visibility of DNA and mechanical stability, respectively. Second, image registration with nanometer resolution requires nanometer-size fiducial markers unrelated to the analyzed molecules.…”

Section: Discussionmentioning

confidence: 99%

“…This method is limited because identification of specific proteins in heterogeneous assemblies depends on distinct structural features, whereas most proteins have a similar globular shape. Molecular recognition in high-resolution SFM images can be achieved by combining SFM with a fluorescence microscope capable of single-fluorescence detection (4)(5)(6). By labeling individual proteins or single DNA molecules, it is possible to distinguish the position of different components in a highly resolved complex.…”

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confidence: 99%

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Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Sánchez¹,

Kertokalio²,

Rossum-Fikkert³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Essential genome transactions, such as homologous recombination, are achieved by concerted and dynamic interactions of multiple protein components with DNA. Which proteins do what and how, will be reflected in their relative arrangements. However, obtaining high-resolution structural information on the variable arrangements of these complex assemblies is a challenge. Here we demonstrate the versatility of a combined total internal reflection fluorescence and scanning force microscope (TIRF-SFM) to pinpoint fluorescently labeled human homologous recombination protein RAD54 interacting with presynaptic (ssDNA) and postsynaptic (dsDNA) human recombinase RAD51 nucleoprotein filaments. Labeled proteins were localized by superresolution imaging on complex structures in the SFM image with high spatial accuracy. We observed some RAD54 at RAD51 filament ends, as expected. More commonly, RAD54 interspersed along RAD51-DNA filaments. RAD54 promotes RAD51-mediated DNA strand exchange and has been described to both stabilize and destabilize RAD51-DNA filaments. The different architectural arrangements we observe for RAD54 with RAD51-DNA filaments may reflect the diverse roles of this protein in homologous recombination.DNA break repair | genome stability | DNA-protein interaction | image registration | single-molecule microscopy T o understand how proteins cooperate to perform elaborate genome transactions, we need to know how they are arranged relative to each other in functional complexes. Determining the structure of such complex and often variable assemblies is a challenge. The scanning force microscope (SFM) is ideal for visualizing DNA-protein assemblies under native conditions at a resolution limited by the radius of curvature of the scanning tip (usually 5-10 nm) (1-3). This method is limited because identification of specific proteins in heterogeneous assemblies depends on distinct structural features, whereas most proteins have a similar globular shape. Molecular recognition in high-resolution SFM images can be achieved by combining SFM with a fluorescence microscope capable of single-fluorescence detection (4-6). By labeling individual proteins or single DNA molecules, it is possible to distinguish the position of different components in a highly resolved complex.The multiprotein complexes of homologous recombination (HR) are an ideal example to establish methods for hybrid microscopy that can be validated on the basis of known structures and used to describe unknown arrangements. HR is a mesoscale DNA rearrangement process achieved by the coordinated action of several proteins and used for DNA repair (7,8). In the core reaction of HR, human recombinase RAD51-DNA filaments search and invade homologous undamaged dsDNA. After strand exchange between the invading ssDNA and its complement, the homologous strand of the duplex is used as template for repair synthesis. Genetic and biochemical evidence show that several mediator proteins, such as RAD54, are required to regulate RAD51 nucleoprotein filament function and pr...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Sánchez¹,

Kertokalio²,

Rossum-Fikkert³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Super-resolution localization techniques may be used to enhance mapping precision. 19,21–24 The data acquired using these techniques lacks the high resolution of DNA sequencing but offers genomic context and is therefore ideal for aiding sequence assembly, when used in combination with DNA sequencing 25–30 as well as analysis of genomic structural variations on the individual chromosome level. 31,32 …”

Section: Dna Sequencing and Optical Mappingmentioning

confidence: 99%

Toward Single-Molecule Optical Mapping of the Epigenome

et al. 2013

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The last decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity on the population level. The complex composition of the genome has made it an ideal system to study on the single-molecule level and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nano-scale and enhanced optical resolving power for analysis of genomic information. In the very last years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

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“…The combination of high resolution fluorescence microscopy with AFM (FIONA-AFM) allows the identification and the localization of fluorescent-tagged molecules such as proteins on topographic image. The strength of this technique was evaluated by the analysis of the interaction of the protein complex UvrA-UvrB, involved in the initiation of nucleotide excision repair pathway, with DNA, and the binding of RNAP on Escherichia coli DNA (Ebenstein et al, 2009;Fronczek et al, 2011). Both studies showed the correlation between optical and AFM signals, providing a proof of principle for combining structural details of multi-molecular complexes with dynamic information yielded by fluorescence.…”

Section: Next Generation Afm For the Study Of Specific Molecules In Bmentioning

confidence: 99%

Atomic Force Microscopy of Chromatin

Quénet¹,

Dimitriadis²,

Dalal³

2012

Atomic Force Microscopy Investigations Into Biology - From Cell to Protein

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Combining atomic force and fluorescence microscopy for analysis of quantum‐dot labeled protein–DNA complexes

Cited by 26 publications

References 24 publications

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51–DNA filaments

Toward Single-Molecule Optical Mapping of the Epigenome

Atomic Force Microscopy of Chromatin

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