Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging

Tanasie, Nicoleta-Loredana; Gutiérrez-Escribano, Pilar; Jaklin, Sigrun; Aragón, Luís; Stigler, Johannes

doi:10.1016/j.celrep.2022.111778

Cited by 15 publications

(14 citation statements)

References 61 publications

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“…The power of integrated dual-beam OT-confocal scanning force-fluorescence spectroscopy measurements has been demonstrated in the investigation of various biological systems. − To make the best use of this powerful tool, careful preparation of biological samples, proper experimental procedures, and rigorous data analysis are all crucial. Here, we have presented a versatile data acquisition and analysis pipeline designed for investigating protein:DNA interactions using an OT-confocal scanning microscope.…”

Section: Discussionmentioning

confidence: 99%

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force–Confocal Fluorescence Microscopy

Liu,

van Veen,

Sánchez

et al. 2024

ACS Photonics

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Integrated single-molecule force−fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force−fluorescence spectroscopy studies on a large variety of biological systems.

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Section: Discussionmentioning

confidence: 99%

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force–Confocal Fluorescence Microscopy

Liu,

van Veen,

Sánchez

et al. 2024

ACS Photonics

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“…Bridging dsDNA with ssDNA could also be sufficient to account for DSB end tethering. Otherwise, cohesin recruitment could be mediated by Smc5/6, which interacts with ssDNA through its hinge domain ( 46, 47 ), and stably associates with ss-dsDNA junctions ( 47, 48 ). Smc5/6, that bears both ubiquitin and SUMO ligase activity, could then locally modify a pool of cohesin, promoting cohesin oligomerization and DSB end-tethering.…”

Section: Discussionmentioning

confidence: 99%

Cohesin complex oligomerization maintains end-tethering at DNA double-strand breaks

Phipps,

Toulouze,

Ducrot

et al. 2023

Preprint

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DNA double-strand breaks (DSB) must be repaired to ensure genome stability. Crucially, DSB ends must be kept together for timely repair. InSaccharomyces cerevisiae, two poorly understood pathways mediate DSB end-tethering. One employs the Mre11-Rad50-Xrs2 (MRX) complex to physically bridge DSB ends. Another requires the conversion of DSB ends into single-strand DNA (ssDNA) by Exo1, but the bridging proteins are unknown. We uncover that cohesin, its loader and Smc5/6 act with Exo1 to tether DSB ends. Remarkably, cohesin specifically impaired in oligomerization fails to tether DSB ends, revealing a new function for cohesin oligomerization. In addition to the known importance of sister chromatid cohesion, microscopy-based microfluidic experiments unveil a new role for cohesin in repair by ensuring DSB end-tethering. Altogether, our findings demonstrate that oligomerization of cohesin prevents DSB end separation and promotes DSB repair, revealing a novel mode of action and role for cohesin in safeguarding genome integrity.

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“…SMC5/6 DNA binding functions are also important. In vitro, purified yeast SMC5/6 binds to ssDNA and dsDNA through different interaction domains [24][25][26] . Recent data demonstrate binding of purified SMC5/6 preferentially to ssDNA-dsDNA junctions, which mimic DNA replication and repair structures 25,26 .…”

Section: Discussionmentioning

confidence: 99%

“…Similar to the related condensin and cohesin SMCs, SMC5/6 interacts with DNA to influence genome stability. Notably, SMC5/6 can bind to both ssDNA and double-stranded (ds)DNA, and can stabilize ssDNA-dsDNA junctions (Chang et al 2022;Tanasie et al 2022). While condensin and cohesin act in chromosome folding and segregation, the function of SMC5/6 in genome maintenance is less well-defined.…”

Section: Introductionmentioning

confidence: 99%

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The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress

O’Leary,

Hansen,

Fingerman

et al. 2023

Preprint

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Mutational patterns consistent with the activity of the APOBEC3 cytidine deaminases are evident in more than half of human cancer genomes. APOBEC3-mediated mutagenesis is genotoxic when uncontrolled due to accumulation of base mutations, replication stress, and DNA breaks. In particular, the APOBEC3A family member is a potent enzyme with nuclear localization that causes substantial DNA damage in experimental systems and human tumors. However, the spectrum of genome-protective mechanisms that ensure genome stability in cells with active APOBEC3A is unknown. Through a genome-wide functional screen, we identify the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is expressed. Cells depleted of SMC5/6 incurred substantial DNA damage when APOBEC3A was active, as reflected by increased DNA breaks, DNA damage signaling, and defective proliferation. We observed an absence of APOBEC3A mutagenesis in human tumors with dysfunction of SMC5/6, consistent with synthetic lethality. APOBEC3A is known to act on ssDNA at replication forks. We observed increased DNA damage in replicating cells in the absence of SMC5/6, suggestive of replication forks as a source of DNA breaks. We interrogated replication fork dynamics by DNA fiber spreading and found a consistent increase in the length of replication tracks upon APOBEC3A activity across multiple cell lines. Increased replication fork length was dependent on Primpol, consistent with a repriming mechanism downstream of APOBEC3A-induced lesions. Loss of SMC5/6 resulted in abrogation of fork elongation in cells with active APOBEC3A, along with increased DNA breaks. Our findings indicate that increased length of replication forks in response to APOBEC3A is a genome-protective response and is dependent on intact SMC5/6. Therefore, SMC5/6 may be a therapeutic vulnerability in tumors in which APOBEC3A is active.

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Stabilization of DNA fork junctions by Smc5/6 complexes revealed by single-molecule imaging

Cited by 15 publications

References 61 publications

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force–Confocal Fluorescence Microscopy

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force–Confocal Fluorescence Microscopy

Cohesin complex oligomerization maintains end-tethering at DNA double-strand breaks

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress

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