Nuclear Folate Metabolism

2018 ◽  
Vol 38 (1) ◽  
pp. 219-243 ◽  
Author(s):  
Martha S. Field ◽  
Elena Kamynina ◽  
James Chon ◽  
Patrick J. Stover
Keyword(s):  
Mammalian Cells ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Childbearing Age ◽  
Definitive Evidence ◽  
Dna Replication And Repair ◽  
One Carbon Metabolism ◽  
Salvage Pathways ◽  
Thymidylate Synthesis

Despite unequivocal evidence that folate deficiency increases risk for human pathologies, and that folic acid intake among women of childbearing age markedly decreases risk for birth defects, definitive evidence for a causal biochemical pathway linking folate to disease and birth defect etiology remains elusive. The de novo and salvage pathways for thymidylate synthesis translocate to the nucleus of mammalian cells during S- and G2/M-phases of the cell cycle and associate with the DNA replication and repair machinery, which limits uracil misincorporation into DNA and genome instability. There is increasing evidence that impairments in nuclear de novo thymidylate synthesis occur in many pathologies resulting from impairments in one-carbon metabolism. Understanding the roles and regulation of nuclear de novo thymidylate synthesis and its relationship to genome stability will increase our understanding of the fundamental mechanisms underlying folate- and vitamin B12–associated pathologies.

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.

scholarly journals Dicer promotes genome stability via the bromodomain transcriptional co-activator Brd4.

2021 ◽  
Author(s):  
Michael Gutbrod ◽  
Benjamin Roche ◽  
Joshua Steinberg ◽  
Asad Lakhani ◽  
Kenneth Chang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Genome Instability ◽  
Embryonic Stem ◽  
Major Satellite ◽  
Suppressor Mutations ◽  
Satellite Repeats ◽  
Transcription And Replication

RNA interference is essential for transcriptional silencing and genome stability, but conservation of this role in mammals has been difficult to demonstrate. Dicer1-/- mouse embryonic stem cells have microRNA-independent proliferation defects, and we conducted a CRISPR-Cas9 screen to restore viability. We identified suppressor mutations in transcriptional activators, H3K9 methyltransferases, and chromosome segregation factors, strongly resembling Dicer suppressors in fission yeast. Suppressors rescued chromosomal defects, and reversed strand-specific transcription of major satellite repeats in Dicer1-/-. The strongest suppressors were in Brd4, and in the transcriptional elongator/histone acetyltransferase Elp3. Using viable mutants and pharmaceutical inhibitors, we demonstrate that deletion of specific residues in Brd4 rescue genome instability defects of Dicer1-/- in both mammalian cells and fission yeast, implicating Dicer in coordinating transcription and replication of satellite repeats.

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.

DNA ligase I null mouse cells show normal DNA repair activity but altered DNA replication and reduced genome stability

2002 ◽  
Vol 115 (7) ◽  
pp. 1551-1561 ◽  
Author(s):  
Darren J. Bentley ◽  
Caroline Harrison ◽  
Ann-Marie Ketchen ◽  
Nicola J. Redhead ◽  
Kay Samuel ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Genome Instability ◽  
Null Alleles ◽  
Dna Ligase ◽  
Null Mouse ◽  
Dna Ligase I ◽  
Mouse Cells

DNA ligase I is the key ligase for DNA replication in mammalian cells and has also been reported to be involved in a number of recombination and repair processes. Our previous finding that Lig1 knockout mouse embryos developed normally to mid-term before succumbing to a specific haematopoietic defect was difficult to reconcile with a report that DNA ligase I is essential for the viability of cultured mammalian cells. To address this issue, we generated a second Lig1 targeted allele and found that the phenotypes of our two Lig1 mutant mouse lines are identical. Widely different levels of Lig1 fusion transcripts were detected from the two targeted alleles, but we could not detect any DNA ligase I protein, and we believe both are effective Lig1 null alleles. Using foetal liver cells to repopulate the haematopoietic system of lethally irradiated adult mice, we demonstrate that the haematopoietic defect in DNA-ligase-I-deficient embryos is a quantitative deficiency relating to reduced proliferation rather than a qualitative block in any haematopoietic lineage. DNA ligase I null fibroblasts from Lig1 mutant embryos showed an accumulation of DNA replication intermediates and increased genome instability. In the absence of a demonstrable deficiency in DNA repair we postulate that, unusually, genome instability may result directly from the DNA replication defect. Lig1null mouse cells performed better in the survival and replication assays than a human LIG1 point mutant, and we suggest that the complete absence of DNA ligase I may make it easier for another ligase to compensate for DNA ligase I deficiency.

scholarly journals Microsatellite instability as a tool to diagnose genome instability in CHO cell culture

2019 ◽  
Author(s):  
Dylan G. Chitwood ◽  
Qinghua Wang ◽  
Kathryn Elliott ◽  
Aiyana Bullock ◽  
Dwon Jordana ◽  
...  
Keyword(s):  
Microsatellite Instability ◽  
Genome Stability ◽  
De Novo ◽  
Genome Instability ◽  
Chinese Hamster ◽  
Loss Of Function ◽  
Cell Numbers ◽  
Functional Regions ◽  
Candidate Loci ◽  
Waste Accumulation

AbstractAs 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 have been due to increasing cell numbers in the bioreactors, and with those increases in cell numbers, strategies have been developed to minimize metabolite waste accumulation, such as lactate and ammonia. Unfortunately, cell growth cannot occur without some waste metabolite accumulation, as central metabolism is required to produce the biopharmaceutical. 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 less than optimal culture environment, little is known about the genotoxic properties and the influence of these compounds on global genome instability. In this study, we examined the effects on Chinese hamster ovary (CHO) cells’ genome sequences and physiology due to exposure to elevated ammonia levels. We identified genome-wide de novo mutations, in addition to variants in functional regions of certain genes involved in the mismatch repair (MMR) pathway, such as DNA2, BRCA1 and RAD52, which led to loss-of-function and eventual genome instability. Additionally, we characterized the presence of microsatellites against the most recent Chinese Hamster genome assembly and discovered certain loci are not replicated faithfully in the presence of elevated ammonia, which represents microsatellite instability (MSI). Furthermore, we found 124 candidate loci that may be suitable biomarkers to gauge genome stability in CHO cultures.

scholarly journals NAD+ Metabolism and Diseases with Motor Dysfunction

Genes ◽  
2021 ◽  
Vol 12 (11) ◽  
pp. 1776
Author(s):  
Samuel Lundt ◽  
Shinghua Ding
Keyword(s):  
Neurodegenerative Diseases ◽  
Motor Neurons ◽  
Mammalian Cells ◽  
De Novo ◽  
Motor Dysfunction ◽  
Salvage Pathway ◽  
Motor Neuron Diseases ◽  
Nad Metabolism ◽  
Charcot Marie Tooth Disease ◽  
Salvage Pathways

Neurodegenerative diseases result in the progressive deterioration of the nervous system, with motor and cognitive impairments being the two most observable problems. Motor dysfunction could be caused by motor neuron diseases (MNDs) characterized by the loss of motor neurons, such as amyotrophic lateral sclerosis and Charcot–Marie–Tooth disease, or other neurodegenerative diseases with the destruction of brain areas that affect movement, such as Parkinson’s disease and Huntington’s disease. Nicotinamide adenine dinucleotide (NAD+) is one of the most abundant metabolites in the human body and is involved with numerous cellular processes, including energy metabolism, circadian clock, and DNA repair. NAD+ can be reversibly oxidized-reduced or directly consumed by NAD+-dependent proteins. NAD+ is synthesized in cells via three different paths: the de novo, Preiss–Handler, or NAD+ salvage pathways, with the salvage pathway being the primary producer of NAD+ in mammalian cells. NAD+ metabolism is being investigated for a role in the development of neurodegenerative diseases. In this review, we discuss cellular NAD+ homeostasis, looking at NAD+ biosynthesis and consumption, with a focus on the NAD+ salvage pathway. Then, we examine the research, including human clinical trials, focused on the involvement of NAD+ in MNDs and other neurodegenerative diseases with motor dysfunction.

scholarly journals Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 530
Author(s):  
Marlo K. Thompson ◽  
Robert W. Sobol ◽  
Aishwarya Prakash
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Gene Editing ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Specific Gene ◽  
Target Sequence ◽  
Transcription Activator ◽  
Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

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 Amazing Acrobat: Yeast’s Histone H3K56 Juggles Several Important Roles While Maintaining Perfect Balance

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 342
Author(s):  
Lihi Gershon ◽  
Martin Kupiec
Keyword(s):  
Dna Replication ◽  
Genome Stability ◽  
Entry And Exit ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cellular Processes ◽  
M Phase ◽  
Dna Replication And Repair ◽  
H3k56 Acetylation ◽  
Timely Fashion ◽  
The Many

Acetylation on lysine 56 of histone H3 of the yeast Saccharomyces cerevisiae has been implicated in many cellular processes that affect genome stability. Despite being the object of much research, the complete scope of the roles played by K56 acetylation is not fully understood even today. The acetylation is put in place at the S-phase of the cell cycle, in order to flag newly synthesized histones that are incorporated during DNA replication. The signal is removed by two redundant deacetylases, Hst3 and Hst4, at the entry to G2/M phase. Its crucial location, at the entry and exit points of the DNA into and out of the nucleosome, makes this a central modification, and dictates that if acetylation and deacetylation are not well concerted and executed in a timely fashion, severe genomic instability arises. In this review, we explore the wealth of information available on the many roles played by H3K56 acetylation and the deacetylases Hst3 and Hst4 in DNA replication and repair.

Sign in / Sign up

Export Citation Format

Share Document