Faculty Opinions recommendation of The homologous recombination machinery modulates the formation of RNA-DNA hybrids and associated chromosome instability.

2013 ◽  
Author(s):  
Michael Lichten
Keyword(s):  
Homologous Recombination ◽  
Chromosome Instability

scholarly journals Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

2021 ◽  
Author(s):  
Thomas R. Reich ◽  
Christian Schwarzenbach ◽  
Juliana Brandstetter Vilar ◽  
Sven Unger ◽  
Fabian Mühlhäusler ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Nuclear Export ◽  
Cell Model ◽  
Chromosome Instability ◽  
Direct Interaction ◽  
High Grade Glioma ◽  
Strand Break ◽  
Γh2ax Foci

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

scholarly journals The homologous recombination machinery modulates the formation of RNA–DNA hybrids and associated chromosome instability

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Lamia Wahba ◽  
Steven K Gore ◽  
Douglas Koshland
Keyword(s):  
Homologous Recombination ◽  
Genome Instability ◽  
Chromosome Instability ◽  
Rna Degradation ◽  
Strand Exchange ◽  
Hybrid Formation ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Yeast Mutants

Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo.

scholarly journals Loss of Cdc13 causes genome instability by a deficiency in replication-dependent telomere capping

2019 ◽  
Author(s):  
Rachel E Langston ◽  
Dominic Palazzola ◽  
Erin Bonnell ◽  
Raymund J. Wellinger ◽  
Ted Weinert
Keyword(s):  
Homologous Recombination ◽  
Genome Stability ◽  
Genome Instability ◽  
Chromosome Instability ◽  
S Phase ◽  
Temperature Sensitive ◽  
End Joining ◽  
Telomere Capping ◽  
Non Homologous End Joining

AbstractIn budding yeast, Cdc13, Stn1, and Ten1 form a telomere binding heterotrimer dubbed CST. Here we investigate the role of Cdc13/CST in maintaining genome stability, using a Chr VII disome system that can generate recombinants, loss, and enigmatic unstable chromosomes. In cells expressing a temperature sensitive CDC13 allele, cdc13F684S, unstable chromosomes frequently arise due to problems in or near a telomere. Hence, when Cdc13 is defective, passage through S phase causes Exo1-dependent ssDNA and unstable chromosomes, which then are the source for whole chromosome instability events (e.g. recombinants, chromosome truncations, dicentrics, and/or loss). Specifically, genome instability arises from a defect in Cdc13’s replication-dependent telomere capping function, not Cdc13s putative post-replication telomere capping function. Furthermore, the unstable chromosomes form without involvement of homologous recombination nor non-homologous end joining. Our data suggest that a Cdc13/CST defect in semi-conservative replication near the telomere leads to ssDNA and unstable chromosomes, which then are lost or subject to complex rearrangements. This system defines a links between replication-dependent chromosome capping and genome stability in the form of unstable chromosomes.

scholarly journals Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

1986 ◽  
Vol 6 (11) ◽  
pp. 3621-3625 ◽  
Author(s):  
M T Boylan ◽  
M J Holland ◽  
W E Timberlake
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Aspergillus Nidulans ◽  
Plasmid Dna ◽  
Chromosome Instability ◽  
Chromosome Stability ◽  
Chromosomal Integration ◽  
Centromeric Dna ◽  
Preferential Loss ◽  
Chromosome Iii

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.

scholarly journals The Mouse Genomic Instability Mutation chaos1 Is an Allele of Polq That Exhibits Genetic Interaction with Atm

2004 ◽  
Vol 24 (23) ◽  
pp. 10381-10389 ◽  
Author(s):  
Naoko Shima ◽  
Robert J. Munroe ◽  
John C. Schimenti
Keyword(s):  
Homologous Recombination ◽  
Genetic Interaction ◽  
Chromosome Instability ◽  
Artificial Chromosome ◽  
Strand Break ◽  
Complementation Test ◽  
Recessive Mutation ◽  
Dna Double Strand Break ◽  
Recombination Pathway

ABSTRACT chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase θ. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority (∼90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.

scholarly journals Inactivation ofUaf1Causes Defective Homologous Recombination and Early Embryonic Lethality in Mice

2013 ◽  
Vol 33 (22) ◽  
pp. 4360-4370 ◽  
Author(s):  
Eunmi Park ◽  
Jung Min Kim ◽  
Benjamin Primack ◽  
David M. Weinstock ◽  
Lisa A. Moreau ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Chromosome Instability ◽  
Embryonic Stem ◽  
Deubiquitinating Enzyme ◽  
Embryonic Fibroblasts ◽  
Early Embryonic Lethality ◽  
Recombination Repair ◽  
Heterodimeric Complex

The deubiquitinating enzyme heterodimeric complex USP1-UAF1 regulates the Fanconi anemia (FA) DNA repair pathway. Absence of this complex leads to increased cellular levels of ubiquitinated FANCD2 (FANCD2-Ub) and ubiquitinated PCNA (PCNA-Ub). Mice deficient in the catalytic subunit of the complex, USP1, exhibit an FA-like phenotype and have a cellular deficiency in homologous-recombination (HR) repair. Here, we have characterized mice deficient in the UAF1 subunit.Uaf1+/−mice were small at birth and exhibited reduced fertility, thus resemblingUsp1−/−mice. Unexpectedly, homozygousUaf1−/−embryos died at embryonic day 7.5 (E7.5). These mutant embryos were small and developmentally retarded. As expected, Uaf1 deficiency in mice led to increased levels of cellular Fancd2-Ub and Pcna-Ub.Uaf1+/−murine embryonic fibroblasts (MEFs) exhibited profound chromosome instability, genotoxin hypersensitivity, and a significant defect in homologous-recombination repair. Moreover,Uaf1−/−mouse embryonic stem cells (mESCs) showed chromosome instability, genotoxin hypersensitivity, and impaired Fancd2 focus assembly. Similar to USP1 knockdown, UAF1 knockdown in tumor cells caused suppression of tumor growthin vivo. Taken together, our data demonstrate the important regulatory role of the USP1-UAF1 complex in HR repair through its regulation of the FANCD2-Ub and PCNA-Ub cellular pools.

scholarly journals RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle

2021 ◽  
Vol 22 (7) ◽  
pp. 3740
Author(s):  
Zuzana Nascakova ◽  
Barbora Boleslavska ◽  
Vaclav Urban ◽  
Anna Oravetzova ◽  
Edita Vlachova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
Chromosome Instability ◽  
Regulation Of Gene Expression ◽  
Protein A ◽  
G1 Phase ◽  
Loop Formation ◽  
Origin Firing ◽  
Dna Strand ◽  
Template Dna

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome

1986 ◽  
Vol 6 (11) ◽  
pp. 3621-3625
Author(s):  
M T Boylan ◽  
M J Holland ◽  
W E Timberlake
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Homologous Recombination ◽  
Aspergillus Nidulans ◽  
Plasmid Dna ◽  
Chromosome Instability ◽  
Chromosome Stability ◽  
Chromosomal Integration ◽  
Centromeric Dna ◽  
Preferential Loss ◽  
Chromosome Iii

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.

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