scholarly journals Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth

2021 ◽  
Author(s):  
Anika Seel ◽  
Francesco Padovani ◽  
Alissa Finster ◽  
Moritz Mayer ◽  
Daniela Bureik ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Cell Growth ◽  
Cell Volume ◽  
Nuclear Dna ◽  
Limiting Factors ◽  
G1 Arrest ◽  
Mitochondrial Dna Copy Number ◽  
Dna Maintenance

AbstractTo maintain stable DNA concentrations, proliferating cells need to coordinate DNA replication with cell growth. For nuclear DNA, eukaryotic cells achieve this by coupling DNA replication to cell cycle progression, ensuring that DNA is doubled exactly once per cell cycle. By contrast, mitochondrial DNA replication is typically not strictly coupled to the cell cycle, leaving the open question of how cells maintain the correct amount of mitochondrial DNA during cell growth. Here, we show that in budding yeast, mitochondrial DNA copy number increases with cell volume, both in asynchronously cycling populations and during G1 arrest. Our findings suggest that cell-volume-dependent mitochondrial DNA maintenance is achieved through nuclear encoded limiting factors, including the mitochondrial DNA polymerase Mip1 and the packaging factor Abf2, whose amount increases in proportion to cell volume. By directly linking mitochondrial DNA maintenance to nuclear protein synthesis, and thus cell growth, constant mitochondrial DNA concentrations can be robustly maintained without a need for cell-cycle-dependent regulation.

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle

1994 ◽  
Vol 107 (12) ◽  
pp. 3515-3520
Author(s):  
S.G. Pasion ◽  
G.W. Brown ◽  
L.M. Brown ◽  
D.S. Ray
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Nuclear Dna ◽  
Protein A ◽  
Mrna Levels ◽  
Gene Encoding ◽  
Genes Encoding

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

scholarly journals Mitochondrial DNA replication: a PrimPol perspective

2017 ◽  
Vol 45 (2) ◽  
pp. 513-529 ◽  
Author(s):  
Laura J. Bailey ◽  
Aidan J. Doherty
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Nuclear Dna ◽  
Dependent Manner ◽  
Catalytic Activities ◽  
Preferential Formation ◽  
Dna Molecule ◽  
Dna Maintenance ◽  
Dna Primers

PrimPol, (primase–polymerase), the most recently identified eukaryotic polymerase, has roles in both nuclear and mitochondrial DNA maintenance. PrimPol is capable of acting as a DNA polymerase, with the ability to extend primers and also bypass a variety of oxidative and photolesions. In addition, PrimPol also functions as a primase, catalysing the preferential formation of DNA primers in a zinc finger-dependent manner. Although PrimPol's catalytic activities have been uncovered in vitro, we still know little about how and why it is targeted to the mitochondrion and what its key roles are in the maintenance of this multicopy DNA molecule. Unlike nuclear DNA, the mammalian mitochondrial genome is circular and the organelle has many unique proteins essential for its maintenance, presenting a differing environment within which PrimPol must function. Here, we discuss what is currently known about the mechanisms of DNA replication in the mitochondrion, the proteins that carry out these processes and how PrimPol is likely to be involved in assisting this vital cellular process.

Wachstumsparameter in normalen und durch Actinomycin verlängerten Zellzyklen von Tetrahymena / Growth Parameters in Normal and Actinomycin-induced prolonged Cell Cycles of Tetrahymena

1969 ◽  
Vol 24 (12) ◽  
pp. 1624-1629 ◽  
Author(s):  
Günter Cleffmann
Keyword(s):  
Cell Cycle ◽  
Protein Synthesis ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Growth ◽  
Rna Synthesis ◽  
Growth Parameters ◽  
Average Duration ◽  
Cell Cycles ◽  
Growing Cell

Actinomycin in low concentration (0,2 μg/ml — 0,5 μg/ml) prolongs the average duration of the cell cycle of Tetrahymena considerably, but does not inhibit cell division completely. Some parameters of the growing cell have been tested in cell cycles extended in this way and compared to those of normally growing cells. The RNA synthesis of treated cells is reduced to such an extent that the RNA content per cell decreases during the prolonged cell cycle. Nevertheless cell growth, protein synthesis and DNA replication proceed at almost the same rate as in untreated cells. These findings indicate that the presence of actinomycin does not interfere with RNA fractions necessary for growth but reduce the synthesis of RNA fractions which are essential for cell division. Therefore a longer period is needed for their accumulation.

scholarly journals Bombyx mori Dihydroorotate Dehydrogenase: Knockdown Inhibits Cell Growth and Proliferation via Inducing Cell Cycle Arrest

2018 ◽  
Vol 19 (9) ◽  
pp. 2581 ◽  
Author(s):  
Erhu Zhao ◽  
Xiaolan Jiang ◽  
Hongjuan Cui
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Dna Replication ◽  
Cell Growth ◽  
Cell Cycle Arrest ◽  
Bombyx Mori ◽  
Dihydroorotate Dehydrogenase ◽  
Pyrimidine Synthesis ◽  
Cycle Arrest ◽  
Cell Growth And Proliferation

Dihydroorotate dehydrogenase (DHODH), in the de novo pyrimidine biosynthetic pathway, is the fourth enzyme of pyrimidine synthesis and is used to oxidize dihydroorotate and hence to orotat. We cloned and characterized here the dhod of silkworms, Bombyx mori. The full-length cDNA sequence of dhod is 1339 bp, including an open reading frame (ORF) of 1173 bp that encoded a 390 amino acid protein. And two domains were involved in the Dihydroorotate dehydrogenase amino acid sequence of silkworms, Bombyx mori (BmDHODH), namely a DHO_dh domain and a transmembrane domain in N-termina. The silkworm dhod is expressed throughout development and in nine tissues. Moreover, knockdown of the silkworm dhod gene reduced cell growth and proliferation through G2/M phase cell cycle arrest. Similarly, DHODH inhibitor (leflunomide) also reduced cell growth and proliferation, with a significant decrease of cyclin B and cdk2. DHODH is the fourth enzyme of pyrimidine synthesis, so we also found that leflunomide can inhibit, at least in part, the endomitotic DNA replication in silk glands cells. These findings demonstrate that downregulation of BmDHODH inhibits cell growth and proliferation in silkworm cells, and the endomitotic DNA replication in silk gland cells.

Induction of G1 Arrest and Apoptosis in a Conditional Expression Model of AML1/ETO: P53-Independent Upregulation of P21/WAF/CIP1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2576-2576
Author(s):  
Tobias Berg ◽  
Manfred Fliegauf ◽  
Jurij Pitako ◽  
Jan Burger ◽  
Mahmoud Abdelkarim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Parp Cleavage ◽  
G1 Arrest ◽  
U937 Cells ◽  
Negative Cell ◽  
Conditional Expression

Abstract Background: The translocation (8;21) is the most common chromosomal rearrangement in AML, resulting in the expression of the fusion protein AML1/ETO. We have developed an ecdysone-inducible U937 model, in which AML1/ETO is expressed in response to treatment with Ponasterone (Pon) A (Fliegauf et al, Oncogene 2004). This model system was used to determine the cellular effects of AML1/ETO and to identify its target genes in U937 cells. Methods: Effects of AML1/ETO expression upon cell growth, viability, cell cycle and apoptosis were analyzed by trypan blue exclusion, FACS analysis using propidium iodide and DiOC6 staining, DNA laddering and Western blot for PARP cleavage, respectively. The gene expression profile of U937 with and without conditional AML1/ETO expression was assessed using Affymetrix U133A microarrays. Wild-type U937 cells with and without PonA treatment as well as AML1/ETO-negative and AML1/ETO-positive myeloid cell lines served as controls. Northern and Western Blotting were used for validation of expression changes. Results: Induction of AML1/ETO expression in U937 resulted in reduced cell growth, G1 arrest and in apoptosis beginning 48–72 hours after PonA treatment. To investigate the underlying mechanisms, microarray analysis was performed. Expression profiles of AML1/ETO-positive and AML1/ETO-negative cell lines formed distinct clusters. Based on stringent criteria, 191 different genes were found upregulated, whereas 37 were downregulated upon expression of AML1/ETO in U937. The identified genes were screened for genes with known functions in cell cycle and apoptosis by automated and manual review and included 13 apoptosis-related genes. Among them, the CDK inhibitor p21/WAF/CIP1 was upregulated 19-fold upon induction of AML1/ETO, whereas the apoptosis regulator MCL-1 was induced 2.5-fold. Based on our criteria, no differential expression of other transcriptionally-controlled apoptosis regulators (such as BCL2, BAX, BAK1, BAD or c-flip) was noted. Northern and Western Blot analysis confirmed the strong induction of p21/WAF/CIP1 that paralleled the expression of AML1/ETO 10 hours after PonA treatment. Induction of p21/WAF/CIP1 was independent of the tumor suppressor protein p53 (Dou et al., Proc. Natl. Acad. Sci. 1995), and by Western blot, p53 was undetectable in U937. Northern Blot analysis revealed a higher expression of p21/WAF/CIP1 in the AML1/ETO-positive cell lines Kasumi-1 and SKNO-1 than in the AML1/ETO-negative cell lines HL-60, KG-1 and U937, supporting our finding that AML1/ETO may induce p21/WAF/CIP1. Conclusions: AML1/ETO expression resulted in increased expression of p21/WAF/CIP1, which might contribute to the observed growth arrest and induction of apoptosis caused by the conditional expression of AML1/ETO.

scholarly journals Human Dna2 Is a Nuclear and Mitochondrial DNA Maintenance Protein

2009 ◽  
Vol 29 (15) ◽  
pp. 4274-4282 ◽  
Author(s):  
Julien P. Duxin ◽  
Benjamin Dao ◽  
Peter Martinsson ◽  
Nina Rajala ◽  
Lionel Guittat ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Telomere Maintenance ◽  
Okazaki Fragment Processing ◽  
Biochemical Fractionation ◽  
Mitochondrial Nucleoids ◽  
Mutant Forms ◽  
Dna Maintenance ◽  
Replication Intermediates ◽  
Chromatin Bridges

ABSTRACT Dna2 is a highly conserved helicase/nuclease that in yeast participates in Okazaki fragment processing, DNA repair, and telomere maintenance. Here, we investigated the biological function of human Dna2 (hDna2). Immunofluorescence and biochemical fractionation studies demonstrated that hDna2 was present in both the nucleus and the mitochondria. Analysis of mitochondrial hDna2 revealed that it colocalized with a subfraction of DNA-containing mitochondrial nucleoids in unperturbed cells. Upon the expression of disease-associated mutant forms of the mitochondrial Twinkle helicase which induce DNA replication pausing/stalling, hDna2 accumulated within nucleoids. RNA interference-mediated depletion of hDna2 led to a modest decrease in mitochondrial DNA replication intermediates and inefficient repair of damaged mitochondrial DNA. Importantly, hDna2 depletion also resulted in the appearance of aneuploid cells and the formation of internuclear chromatin bridges, indicating that nuclear hDna2 plays a role in genomic DNA stability. Together, our data indicate that hDna2 is similar to its yeast counterpart and is a new addition to the growing list of proteins that participate in both nuclear and mitochondrial DNA maintenance.

scholarly journals SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

1973 ◽  
Vol 58 (2) ◽  
pp. 340-345 ◽  
Author(s):  
Kenneth D. Ley ◽  
Marilyn M. Murphy
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Time Course ◽  
Nuclear Dna ◽  
Tritiated Thymidine ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
In Cells

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

scholarly journals Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication

2015 ◽  
Vol 112 (30) ◽  
pp. 9334-9339 ◽  
Author(s):  
J. Bradley Holmes ◽  
Gokhan Akman ◽  
Stuart R. Wood ◽  
Kiran Sakhuja ◽  
Susana M. Cerritelli ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Dna Replication ◽  
Strand Break ◽  
Initiation Site ◽  
Major Noncoding Region ◽  
Mitochondrial Dna Replication ◽  
Strand Initiation ◽  
Mitochondrial Dna Polymerase ◽  
Dna Maintenance

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

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