scholarly journals THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

2021 ◽  
Author(s):  
Rebecca E. Brown ◽  
Xiaofeng A. Su ◽  
Stacey Fair ◽  
Katherine Wu ◽  
Lauren Verra ◽  
...  
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Repetitive Sequences ◽  
Rna Degradation ◽  
Cag Repeat ◽  
Cag Repeats ◽  
Dna Breaks ◽  
Complementary Role ◽  
Rna Export ◽  
Tramp Complex

AbstractExpansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

scholarly journals Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability

2019 ◽  
Vol 116 (36) ◽  
pp. 18021-18030 ◽  
Author(s):  
Jiajia Tang ◽  
Giada Frascaroli ◽  
Robert J. Lebbink ◽  
Eleonore Ostermann ◽  
Wolfram Brune
Keyword(s):  
Dna Damage ◽  
Cell Fusion ◽  
Human Cytomegalovirus ◽  
Viral Entry ◽  
Genome Stability ◽  
Genome Instability ◽  
Glycoprotein B ◽  
Dna Breaks ◽  
Signaling Axis ◽  
Caspase 2

Human cytomegalovirus (HCMV), like many other DNA viruses, can cause genome instability and activate a DNA damage response (DDR). Activation of ataxia-telangiectasia mutated (ATM), a kinase activated by DNA breaks, is a hallmark of the HCMV-induced DDR. Here we investigated the activation of caspase-2, an initiator caspase activated in response to DNA damage and supernumerary centrosomes. Of 7 HCMV strains tested, only strain AD169 activated caspase-2 in infected fibroblasts. Treatment with an ATM inhibitor or inactivation of PIDD or RAIDD inhibited caspase-2 activation, indicating that caspase-2 was activated by the PIDDosome. A set of chimeric HCMV strains was used to identify the genetic basis of this phenotype. Surprisingly, we found a single nucleotide polymorphism within the AD169 UL55 ORF, resulting in a D275Y amino acid exchange within glycoprotein B (gB), to be responsible for caspase-2 activation. As gB is an envelope glycoprotein required for fusion with host cell membranes, we tested whether gB(275Y) altered viral entry into fibroblasts. While entry of AD169 expressing gB(275D) proceeded slowly and could be blocked by a macropinocytosis inhibitor, entry of wild-type AD169 expressing gB(275Y) proceeded more rapidly, presumably by envelope fusion with the plasma membrane. Moreover, gB(275Y) caused the formation of syncytia with numerous centrosomes, suggesting that cell fusion triggered caspase-2 activation. These results suggest that gB variants with increased fusogenicity accelerate viral entry, cause cell fusion, and thereby compromise genome stability. They further suggest the ATM-PIDDosome-caspase-2 signaling axis alerts the cell of potentially dangerous cell fusion.

scholarly journals SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences

2017 ◽  
Vol 114 (3) ◽  
pp. 552-557 ◽  
Author(s):  
Eric J. Foss ◽  
Uyen Lao ◽  
Emily Dalrymple ◽  
Robin L. Adrianse ◽  
Taylor Loe ◽  
...  
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Repetitive Sequences ◽  
Chromatin Accessibility ◽  
Genome Replication ◽  
Replication Origins ◽  
Experimental Identification

Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age- and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age- and disease-associated genome instability.

scholarly journals MRG15-mediated tethering of PALB2 to unperturbed chromatin protects active genes from genotoxic stress

2017 ◽  
Vol 114 (29) ◽  
pp. 7671-7676 ◽  
Author(s):  
Jean-Yves Bleuyard ◽  
Marjorie Fournier ◽  
Ryuichiro Nakato ◽  
Anthony M. Couturier ◽  
Yuki Katou ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Genome Instability ◽  
Genotoxic Stress ◽  
Missense Mutations ◽  
Dna Breaks ◽  
Metaphase Chromosomes ◽  
Repair Factor ◽  
Active Genes

The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.

scholarly journals RPA-mediated recruitment of Bre1 couples histone H2B ubiquitination to DNA replication and repair

2021 ◽  
Vol 118 (8) ◽  
pp. e2017497118
Author(s):  
Guangxue Liu ◽  
Jiaqi Yan ◽  
Xuejie Wang ◽  
Junliang Chen ◽  
Xin Wang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Genome Instability ◽  
E3 Ligase ◽  
Dna Breaks ◽  
Ubiquitin E3 Ligase ◽  
Dna Replication And Repair ◽  
Local Enrichment

The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome instability and cancer. How the Bre1–H2Bub pathway is evoked in response to DNA replication or repair remains unknown. Here, we identify that the single-stranded DNA (ssDNA) binding factor RPA acts as a key mediator that couples Bre1-mediated H2Bub to DNA replication and repair in yeast. We found that RPA interacts with Bre1 in vitro and in vivo, and this interaction is stimulated by ssDNA. This association ensures the recruitment of Bre1 to replication forks or DNA breaks but does not affect its E3 ligase activity. Disruption of the interaction abolishes the local enrichment of H2Bub, resulting in impaired DNA replication, response to replication stress, and repair by homologous recombination, accompanied by increased genome instability and DNA damage sensitivity. Notably, we found that RNF20, the human homolog of Bre1, interacts with RPA70 in a conserved mode. Thus, RPA functions as a master regulator for the spatial–temporal control of H2Bub chromatin landscape during DNA replication and recombination, extending the versatile roles of RPA in guarding genome stability.

scholarly journals RAD51 promotes non-homologous genetic rearrangements that are prevented by 53BP1

2019 ◽  
Author(s):  
Josée Guirouilh-Barbat ◽  
Wei Yu ◽  
Loelia Babin ◽  
Elisa Yaniz Galende ◽  
Tatiana Popova ◽  
...  
Keyword(s):  
Sequence Homology ◽  
Genetic Instability ◽  
Genome Stability ◽  
Genome Instability ◽  
Homology Search ◽  
Dna Breaks ◽  
Sequence Homologies ◽  
Intrinsic Capacity ◽  
Chromosomal Sequences ◽  
Genetic Rearrangements

AbstractHomologous recombination (HR), which requires long sequence homologies, is considered a high fidelity mechanism, preserving genome stability. In contrast, we show here that the central HR players RAD51 or BRCA2, promote genetic instability, fostering translocations and capture of ectopic chromosomal sequences when joining distant DNA breaks. Surprisingly, these events do not involve sequence homologies. Moreover, our data reveal that 53BP1 protects against RAD51-mediated non-homologous genetic rearrangements. Finally, analysis of a large panel of breast tumors revealed that BRCA2 proficiency is associated with increased frequency of capture of non-homologous sequences at junctions of structural variants (translocations, duplications, inversions, deletions). These data reveal that HR proteins (RAD51, BRCA2) possess the intrinsic capacity to generate genetic instability through sequence homology-independent processes, and that 53BP1 protects against it. We propose that BRCA2/RAD51-mediated genome instability occurs in the course of sequence homology search for HR.

scholarly journals To Repeat or Not to Repeat: Repetitive Sequences Regulate Genome Stability in Candida albicans

Genes ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 866 ◽  
Author(s):  
Dunn ◽  
Anderson
Keyword(s):  
Candida Albicans ◽  
Genome Stability ◽  
Genome Instability ◽  
Repetitive Sequences ◽  
Gene Families ◽  
Rdna Locus ◽  
Repeat Sequence ◽  
Sequence Evolution ◽  
Adaptive Potential

Genome instability often leads to cell death but can also give rise to innovative genotypic and phenotypic variation through mutation and structural rearrangements. Repetitive sequences and chromatin architecture in particular are critical modulators of recombination and mutability. In Candida albicans, four major classes of repeats exist in the genome: telomeres, subtelomeres, the major repeat sequence (MRS), and the ribosomal DNA (rDNA) locus. Characterization of these loci has revealed how their structure contributes to recombination and either promotes or restricts sequence evolution. The mechanisms of recombination that give rise to genome instability are known for some of these regions, whereas others are generally unexplored. More recent work has revealed additional repetitive elements, including expanded gene families and centromeric repeats that facilitate recombination and genetic innovation. Together, the repeats facilitate C. albicans evolution through construction of novel genotypes that underlie C. albicans adaptive potential and promote persistence across its human host.

scholarly journals Targeting Genome Stability in Melanoma—A New Approach to an Old Field

2021 ◽  
Vol 22 (7) ◽  
pp. 3485
Author(s):  
Marta Osrodek ◽  
Michal Wozniak
Keyword(s):  
Genome Stability ◽  
Genome Instability ◽  
Checkpoint Inhibitors ◽  
Mapk Pathway ◽  
Rapid Development ◽  
Melanoma Cells ◽  
Old Field ◽  
Number Of Patients ◽  
Recent Developments ◽  
Mapk Inhibitors

Despite recent groundbreaking advances in the treatment of cutaneous melanoma, it remains one of the most treatment-resistant malignancies. Due to resistance to conventional chemotherapy, the therapeutic focus has shifted away from aiming at melanoma genome stability in favor of molecularly targeted therapies. Inhibitors of the RAS/RAF/MEK/ERK (MAPK) pathway significantly slow disease progression. However, long-term clinical benefit is rare due to rapid development of drug resistance. In contrast, immune checkpoint inhibitors provide exceptionally durable responses, but only in a limited number of patients. It has been increasingly recognized that melanoma cells rely on efficient DNA repair for survival upon drug treatment, and that genome instability increases the efficacy of both MAPK inhibitors and immunotherapy. In this review, we discuss recent developments in the field of melanoma research which indicate that targeting genome stability of melanoma cells may serve as a powerful strategy to maximize the efficacy of currently available therapeutics.

scholarly journals The Effect of CAG Repeats within the Non-Pathological Range in the HTT Gene on Cognitive Functions in Patients with Subjective Cognitive Decline and Mild Cognitive Impairment

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1051
Author(s):  
Valentina Bessi ◽  
Salvatore Mazzeo ◽  
Silvia Bagnoli ◽  
Giulia Giacomucci ◽  
Assunta Ingannato ◽  
...  
Keyword(s):  
Cognitive Impairment ◽  
Mild Cognitive Impairment ◽  
Cognitive Decline ◽  
Spatial Ability ◽  
Cognitive Status ◽  
Cag Repeat ◽  
Cag Repeats ◽  
Subjective Cognitive Decline ◽  
Visual Spatial ◽  
Premorbid Intelligence

The Huntingtin gene (HTT) is within a class of genes containing a key region of CAG repeats. When expanded beyond 39 repeats, Huntington disease (HD) develops. Individuals with less than 35 repeats are not associated with HD. Increasing evidence has suggested that CAG repeats play a role in modulating brain development and brain function. However, very few studies have investigated the effect of CAG repeats in the non-pathological range on cognitive performances in non-demented individuals. In this study, we aimed to test how CAG repeats’ length influences neuropsychological scores in patients with subjective cognitive decline (SCD) and mild cognitive impairment (MCI). We included 75 patients (46 SCD and 29 MCI). All patients underwent an extensive neuropsychological battery and analysis of HTT alleles to quantify the number of CAG repeats. Results: CAG repeat number was positively correlated with scores of tests assessing for executive function, visual–spatial ability, and memory in SCD patients, while in MCI patients, it was inversely correlated with scores of visual–spatial ability and premorbid intelligence. When we performed a multiple regression analysis, we found that these relationships still remained, also when adjusting for possible confounding factors. Interestingly, logarithmic models better described the associations between CAG repeats and neuropsychological scores. CAG repeats in the HTT gene within the non-pathological range influenced neuropsychological performances depending on global cognitive status. The logarithmic model suggested that the positive effect of CAG repeats in SCD patients decreases as the number of repeats grows.

scholarly journals Characterization of metabolic responses, genetic variations, and microsatellite instability in ammonia-stressed CHO cells grown in fed-batch cultures

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Dylan G. Chitwood ◽  
Qinghua Wang ◽  
Kathryn Elliott ◽  
Aiyana Bullock ◽  
Dwon Jordana ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Microsatellite Loci ◽  
Cho Cells ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Chinese Hamster ◽  
Functional Genes ◽  
Nucleotide Polymorphisms ◽  
De Novo Mutations

Abstract Background As bioprocess intensification has increased over the last 30 years, yields from mammalian cell processes have increased from 10’s of milligrams to over 10’s of grams per liter. Most of these gains in productivity can be attributed to increasing cell densities within bioreactors. As such, strategies have been developed to minimize accumulation of metabolic wastes, such as lactate and ammonia. Unfortunately, neither cell growth nor biopharmaceutical production can occur without some waste metabolite accumulation. Inevitably, metabolic waste accumulation leads to decline and termination of the culture. While it is understood that the accumulation of these unwanted compounds imparts a suboptimal culture environment, little is known about the genotoxic properties of these compounds that may lead to global genome instability. In this study, we examined the effects of high and moderate extracellular ammonia on the physiology and genomic integrity of Chinese hamster ovary (CHO) cells. Results Through whole genome sequencing, we discovered 2394 variant sites within functional genes comprised of both single nucleotide polymorphisms and insertion/deletion mutations as a result of ammonia stress with high or moderate impact on functional genes. Furthermore, several of these de novo mutations were found in genes whose functions are to maintain genome stability, such as Tp53, Tnfsf11, Brca1, as well as Nfkb1. Furthermore, we characterized microsatellite content of the cultures using the CriGri-PICR Chinese hamster genome assembly and discovered an abundance of microsatellite loci that are not replicated faithfully in the ammonia-stressed cultures. Unfaithful replication of these loci is a signature of microsatellite instability. With rigorous filtering, we found 124 candidate microsatellite loci that may be suitable for further investigation to determine whether these loci may be reliable biomarkers to predict genome instability in CHO cultures. Conclusion This study advances our knowledge with regards to the effects of ammonia accumulation on CHO cell culture performance by identifying ammonia-sensitive genes linked to genome stability and lays the foundation for the development of a new diagnostic tool for assessing genome stability.

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