scholarly journals Rad51 paralog complex Rad55–Rad57 acts as a molecular chaperone during homologous recombination

2020 ◽  
Author(s):  
Upasana Roy ◽  
Youngho Kwon ◽  
Lea Marie ◽  
Lorraine Symington ◽  
Patrick Sung ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Cancer Risk ◽  
Single Molecule ◽  
Molecular Chaperone ◽  
Bound States ◽  
Current Model ◽  
Genome Integrity ◽  
Single Molecule Imaging ◽  
Transient Interactions

SummaryHomologous recombination (HR) is essential for the maintenance of genome integrity. Rad51 paralogs fulfill a conserved, but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single–molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57 promotes the assembly of Rad51 recombinase filaments through transient interactions, providing evidence that it acts as a classical molecular chaperone. Srs2 is an ATP–dependent anti–recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55– Rad57 does not physically block the movement of Srs2. Instead, Rad55–Rad57 promotes rapid re– assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and ssDNA–bound states, the rate of which is dynamically controlled though the opposing actions of Rad55–Rad57 and Srs2.

scholarly journals The Role of the Rad55–Rad57 Complex in DNA Repair

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1390
Author(s):  
Upasana Roy ◽  
Eric C. Greene
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Single Molecule ◽  
Genome Integrity ◽  
Genetic Studies ◽  
Biochemical Assays ◽  
Rad51 Paralogs ◽  
Higher Eukaryotes

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55–Rad57 in HR.

scholarly journals Relocation of rDNA repeats for repair is dependent on SUMO-mediated nucleolar release by the Cdc48/p97 segregase

2021 ◽  
Author(s):  
Matías Capella ◽  
Imke K. Mandemaker ◽  
Lucía Martín Caballero ◽  
Boris Pfander ◽  
Andreas G. Ladurner ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Ribosomal Rna ◽  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Genome Integrity ◽  
Repetitive Nature ◽  
Active Transcription ◽  
Rna Genes

AbstractRibosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to active transcription and their repetitive nature. Compartmentalization of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be released to the nucleoplasm to allow repair by homologous recombination. The process of rDNA relocation is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP component Nur1, releasing nuclear membrane-tethered rDNA repeats from the nucleolus in Saccharomyces cerevisiae. Cooperating with Nur1 phosphorylation, SUMOylation targets the rDNA tethering complex for disassembly mediated by the segregase Cdc48/p97, which recognizes SUMOylated CLIP-cohibin through its cofactor, Ufd1. Consistent with the conservation of this mechanism, UFD1L depletion impairs rDNA release in human cells. The dynamic and regulated assembly and disassembly of the CLIP-cohibin complex is therefore a key, conserved determinant of nucleolar rDNA release and genome integrity.

scholarly journals Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matías Capella ◽  
Imke K. Mandemaker ◽  
Lucía Martín Caballero ◽  
Fabian den Brave ◽  
Boris Pfander ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Ribosomal Rna ◽  
Molecular Mechanisms ◽  
Human Cells ◽  
Ribosomal Rna Genes ◽  
Genome Integrity ◽  
Repetitive Nature ◽  
Rna Genes

AbstractRibosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

scholarly journals Single molecule imaging of confomational changes in Cytochrome c frin Saccharomyces cerevisiae

1999 ◽  
Vol 39 (supplement) ◽  
pp. S153
Author(s):  
T. Nishida ◽  
T. Wazawa ◽  
Y. Ishii ◽  
Y. Goto ◽  
T. Yanagida
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cytochrome C ◽  
Single Molecule ◽  
Single Molecule Imaging

scholarly journals A genome-wide screen for genes affecting spontaneous direct-repeat recombination in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Daniele Novarina ◽  
Ridhdhi Desai ◽  
Jessica A. Vaisica ◽  
Jiongwen Ou ◽  
Mohammed Bellaoui ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
High Throughput ◽  
Low Frequency ◽  
Direct Repeat ◽  
Genome Integrity ◽  
Replica Plating ◽  
A Genome ◽  
Mammalian Genomes ◽  
Rnase H2

ABSTRACTHomologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionary conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

scholarly journals A Genome-Wide Screen for Genes Affecting Spontaneous Direct-Repeat Recombination in Saccharomyces cerevisiae

2020 ◽  
Vol 10 (6) ◽  
pp. 1853-1867
Author(s):  
Daniele Novarina ◽  
Ridhdhi Desai ◽  
Jessica A. Vaisica ◽  
Jiongwen Ou ◽  
Mohammed Bellaoui ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
High Throughput ◽  
Low Frequency ◽  
Direct Repeat ◽  
Genome Integrity ◽  
Link Type ◽  
A Genome ◽  
Mammalian Genomes ◽  
Rnase H2

Homologous recombination is an important mechanism for genome integrity maintenance, and several homologous recombination genes are mutated in various cancers and cancer-prone syndromes. However, since in some cases homologous recombination can lead to mutagenic outcomes, this pathway must be tightly regulated, and mitotic hyper-recombination is a hallmark of genomic instability. We performed two screens in Saccharomyces cerevisiae for genes that, when deleted, cause hyper-recombination between direct repeats. One was performed with the classical patch and replica-plating method. The other was performed with a high-throughput replica-pinning technique that was designed to detect low-frequency events. This approach allowed us to validate the high-throughput replica-pinning methodology independently of the replicative aging context in which it was developed. Furthermore, by combining the two approaches, we were able to identify and validate 35 genes whose deletion causes elevated spontaneous direct-repeat recombination. Among these are mismatch repair genes, the Sgs1-Top3-Rmi1 complex, the RNase H2 complex, genes involved in the oxidative stress response, and a number of other DNA replication, repair and recombination genes. Since several of our hits are evolutionarily conserved, and repeated elements constitute a significant fraction of mammalian genomes, our work might be relevant for understanding genome integrity maintenance in humans.

Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

2020 ◽  
Author(s):  
Nikolas Hundt
Keyword(s):  
Single Molecule ◽  
Cellular Systems ◽  
Single Molecule Imaging ◽  
Label Free ◽  
Measurement Sensitivity ◽  
Mass Distributions ◽  
Quantitative Measurements ◽  
Contrast Mechanism ◽  
Cellular Components ◽  
Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

Sign in / Sign up

Export Citation Format

Share Document