scholarly journals Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae

Mutagenesis ◽  
2013 ◽  
Vol 28 (2) ◽  
pp. 187-195 ◽  
Author(s):  
L. Spugnesi ◽  
C. Balia ◽  
A. Collavoli ◽  
E. Falaschi ◽  
V. Quercioli ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Nuclear Foci

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 Saccharomyces cerevisiae H2A copies differentially contribute to recombination and CAG/CTG repeat maintenance, with a role for H2A.1 threonine 126

2018 ◽  
Author(s):  
Nealia C.M. House ◽  
Erica J. Polleys ◽  
Ishtiaque Quasem ◽  
Cailin E. Joyce ◽  
Oliver Takacsi-Nagy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Cag Repeat ◽  
Break Induced Replication ◽  
Sister Chromatid Recombination ◽  
True Homolog ◽  
Ctg Repeat

AbstractDNA are sites of genomic instability. Long CAG/CTG repeats form hairpin structures, are fragile, and can expand during DNA repair. The chromatin response to DNA damage can influence repair fidelity, but the knowledge of chromatin modifications involved in maintaining repair fidelity within repetitive DNA is limited. In a screen for CAG repeat fragility in Saccharomyces cerevisiae, histone 2A copy 1 (H2A.1) was identified to protect the repeat from increased rates of breakage. To address the role of H2A in CAG repeat instability, we tested the effect of deleting each histone H2 subytpe. Whereas deletion of HTA2, HTZ1, HTB1, and HTB2 did not significantly affect CAG repeat maintenance, deletion of HTA1 resulted in increased expansion frequency. Notably, mutation of threonine 126, unique to H2A.1, to a non-phosphorylatable alanine increased CAG repeat instability to a similar level as the hta1Δ mutant. CAG instability in the absence of HTA1 or mutation to hta1-T126A was dependent on the presence of the homologous recombination (HR) repair proteins Rad51, Rad52, and Rad57, and the Polδ subunit Pol32. In addition, sister chromatid recombination (SCR) was suppressed in the hta1Δ and hta1-T126A mutants and this suppression was epistatic to pol32Δ. Finally, break-induced replication (BIR) is impaired in the hta1Δ mutant, resulting in an altered repair profile. These data reveal differential roles for the H2A subtypes in DNA repair and implicate a new role for H2A.1 threonine-126 phosphorylation in mediating fidelity during HR repair and promoting SCR. Using a fragile, repetive DNA element to model endogenous DNA damage, our results demonstrate that H2A.1 plays a greater role than H2A.2 in promoting homology-dependent repair, suggesting H2A.1 is the true homolog of mammalian H2AX, whereas H2A.2 is functionally equivalent to mammalian H2A.Author SummaryCAG/CTG trinuncleotide repeats are fragile sequences that when expanded can cause human disease. To evaluate the role of S. cerevisiae histone H2A copies in DNA repair, we have measured instability of an expanded CAG/CTG repeat tract and repair outcomes in H2A mutants. Although the two copies of H2A are nearly identical in amino acid sequence, we found that the CAG repeat is more unstable in the absence of H2A copy 1 (H2A.1) than H2A copy 2, and that this role appears to be partially dependent on a phosphorylatable threonine at residue 126 in the C-terminal tail of H2A.1. Further, we show through a series of genetic assays that H2A.1 plays a role in promoting homologous recombination events, including sister chromatid recombination and break-induced replication. Our results uncover a role for H2A.1 in mediating fidelity of repair within repetitive DNA, and demonstrate that modification of its unique Thr126 residue plays a role in regulating SCR. Given the dependence of HR repair on H2A.1 but not H2A.2, we conclude that H2A.1 plays a greater repair-specific role in the cell and therefore would be the true homolog of mammalian H2AX.

scholarly journals DNA Damage-Inducible and RAD52-Independent Repair of DNA Double-Strand Breaks in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1085-1099 ◽  
Author(s):  
Carol Wood Moore ◽  
Judith McKoy ◽  
Michelle Dardalhon ◽  
Darline Davermann ◽  
Marcia Martinez ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Homologous Recombination ◽  
Double Strand Breaks ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Γ Irradiation ◽  
Strand Breaks ◽  
Mutant Strains

Abstract Chromosomal repair was studied in stationary-phase Saccharomyces cerevisiae, including rad52/rad52 mutant strains deficient in repairing double-strand breaks (DSBs) by homologous recombination. Mutant strains suffered more chromosomal fragmentation than RAD52/RAD52 strains after treatments with cobalt-60 γ irradiation or radiomimetic bleomycin, except after high bleomycin doses when chromosomes from rad52/rad52 strains contained fewer DSBs than chromosomes from RAD52/RAD52 strains. DNAs from both genotypes exhibited quick rejoining following γ irradiation and sedimentation in isokinetic alkaline sucrose gradients, but only chromosomes from RAD52/RAD52 strains exhibited slower rejoining (10 min to 4 hr in growth medium). Chromosomal DSBs introduced by γ irradiation and bleomycin were analyzed after pulsed-field gel electrophoresis. After equitoxic damage by both DNA-damaging agents, chromosomes in rad52/rad52 cells were reconstructed under nongrowth conditions [liquid holding (LH)]. Up to 100% of DSBs were eliminated and survival increased in RAD52/RAD52 and rad52/rad52 strains. After low doses, chromosomes were sometimes degraded and reconstructed during LH. Chromosomal reconstruction in rad52/rad52 strains was dose dependent after γ irradiation, but greater after high, rather than low, bleomycin doses with or without LH. These results suggest that a threshold of DSBs is the requisite signal for DNA-damage-inducible repair, and that nonhomologous end-joining repair or another repair function is a dominant mechanism in S. cerevisiae when homologous recombination is impaired.

scholarly journals The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

2021 ◽  
Author(s):  
Orsolya Frittmann ◽  
Vamsi K Gali ◽  
Miklos Halmai ◽  
Robert Toth ◽  
Zsuzsanna Gyorfy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
Dna Lesions ◽  
Template Switching ◽  
Adjacent Region ◽  
Yeast Two Hybrid ◽  
Replication Complex ◽  
Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

scholarly journals RAP80 and BRCA1 PARsylation protect chromosome integrity by preventing retention of BRCA1-B/C complexes in DNA repair foci

2020 ◽  
Vol 117 (4) ◽  
pp. 2084-2091
Author(s):  
Jekaterina Vohhodina ◽  
Kimberly J. Toomire ◽  
Sarah A. Petit ◽  
Goran Micevic ◽  
Geeta Kumari ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Complex Formation ◽  
Illegitimate Recombination ◽  
Adp Ribosylation ◽  
Chromosomal Disorder ◽  
Nuclear Foci ◽  
Simultaneous Loss ◽  
Chromosome Integrity ◽  
Focal Structures

BRCA1 promotes error-free, homologous recombination-mediated repair (HRR) of DNA double-stranded breaks (DSBs). When excessive and uncontrolled, BRCA1 HRR activity promotes illegitimate recombination and genome disorder. We and others have observed that the BRCA1-associated protein RAP80 recruits BRCA1 to postdamage nuclear foci, and these chromatin structures then restrict the amplitude of BRCA1-driven HRR. What remains unclear is how this process is regulated. Here we report that both BRCA1 poly-ADP ribosylation (PARsylation) and the presence of BRCA1-bound RAP80 are critical for the normal interaction of BRCA1 with some of its partners (e.g., CtIP and BACH1) that are also known components of the aforementioned focal structures. Surprisingly, the simultaneous loss of RAP80 and failure therein of BRCA1 PARsylation results in the dysregulated accumulation in these foci of BRCA1 complexes. This in turn is associated with the intracellular development of a state of hyper-recombination and gross chromosomal disorder. Thus, physiological RAP80-BRCA1 complex formation and BRCA1 PARsylation contribute to the kinetics by which BRCA1 HRR-sustaining complexes normally concentrate in nuclear foci. These events likely contribute to aneuploidy suppression.

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