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.

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 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 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 A genome-wide resource for high-throughput genomic tagging of yeast ORFs

2017 ◽  
Author(s):  
Matthias Meurer ◽  
Yuanqiang Duan ◽  
Ehud Sass ◽  
Ilia Kats ◽  
Konrad Herbst ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Expression ◽  
High Throughput ◽  
Fluorescent Proteins ◽  
Regulatory Elements ◽  
Targeted Sequencing ◽  
Yeast Proteome ◽  
Genome Wide ◽  
A Genome

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae ORFs. It consists of 5661 strains with an acceptor module inserted after each ORF, which can be efficiently replaced with tags or regulatory elements. We validate the library with targeted sequencing and demonstrate its use by tagging the yeast proteome with bright fluorescent proteins, determining how sequences downstream of ORFs influence protein expression and localizing previously undetected proteins.

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