scholarly journals A double-strand break within a yeast artificial chromosome (YAC) containing human DNA can result in YAC loss, deletion or cell lethality.

1996 ◽  
Vol 16 (8) ◽  
pp. 4414-4425 ◽  
Author(s):  
C B Bennett ◽  
T J Westmoreland ◽  
J R Snipe ◽  
M A Resnick
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Yeast Artificial Chromosome ◽  
Genetic Material ◽  
Artificial Chromosome ◽  
Strand Break ◽  
Double Strand Break ◽  
Chromosomal Dna ◽  
Cell Cycle Delay ◽  
Human Dna

Human chromosomal DNA contains many repeats which might provide opportunities for DNA repair. We have examined the consequences of a single double-strand break (DSB) within a 360-kb dispensable yeast artificial chromosome (YAC) containing human DNA (YAC12). An Alu-URA3-YZ sequence was targeted to several Alu sites within the YAC in strains of the yeast Saccharomyces cerevisiae; the strains contained a galactose-inducible HO endonuclease that cut the YAC at the YZ site. The presence of a DSB in most YACs led to deletion of the URA3 cassette, with retention of the telomeric markers, through recombination between surrounding Alus. For two YACs, the DSBs were not repaired and there was a G2 delay associated with the persistent DSBs. The presence of persistent DSBs resulted in cell death even though the YACs were dispensable. Among the survivors of the persistent DSBs, most had lost the YAC. By a pullback procedure, cell death was observed to begin at least 6 h after induction of a break. For YACs in which the DSB was rapidly repaired, the breaks did not cause cell cycle delay or lead to cell death. These results are consistent with our previous conclusion that a persistent DSB in a plasmid (YZ-CEN) also caused lethality (C. B. Bennett, A. L. Lewis, K. K. Baldwin, and M. A. Resnick, Proc. Natl. Acad. Sci. USA 90:5613-5617, 1993). However, a break in the YZ-CEN plasmid did not induce lethality in the strain (CBY) background used in the present study. The differences in survival levels appear to be due to the rapid degradation of the plasmid in the CBY strain. We, therefore, propose that for a DSB to cause cell cycle delay and death by means other than the loss of essential genetic material, it must remain unrepaired and be long-lived.

Antitumor Effect of Pomolic Acid in Acute Myeloid Leukemia Cells Involves Cell Death, Decreased Cell Growth and Topoisomerases Inhibition

2019 ◽  
Vol 18 (10) ◽  
pp. 1457-1468
Author(s):  
Michelle X.G. Pereira ◽  
Amanda S.O. Hammes ◽  
Flavia C. Vasconcelos ◽  
Aline R. Pozzo ◽  
Thaís H. Pereira ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Natural Compound ◽  
Dna Topoisomerases ◽  
Human Dna ◽  
Pomolic Acid ◽  
Acute Myeloid

Background: Acute myeloid leukemia (AML) represents the largest number of annual deaths from hematologic malignancy. In the United States, it was estimated that 21.380 individuals would be diagnosed with AML and 49.5% of patients would die in 2017. Therefore, the search for novel compounds capable of increasing the overall survival rate to the treatment of AML cells is urgent. Objectives: To investigate the cytotoxicity effect of the natural compound pomolic acid (PA) and to explore the mechanism of action of PA in AML cell lines with different phenotypes. Methods: Three different AML cell lines, HL60, U937 and Kasumi-1 cells with different mechanisms of resistance were used to analyze the effect of PA on the cell cycle progression, on DNA intercalation and on human DNA topoisomerases (hTopo I and IIα) in vitro studies. Theoretical experiments of the inhibition of hTopo I and IIα were done to explore the binding modes of PA. Results: PA reduced cell viability, induced cell death, increased sub-G0/G1 accumulation and activated caspases pathway in all cell lines, altered the cell cycle distribution and inhibited the catalytic activity of both human DNA topoisomerases. Conclusion: Finally, this study showed that PA has powerful antitumor activity against AML cells, suggesting that this natural compound might be a potent antineoplastic agent to improve the treatment scheme of this neoplasm.

scholarly journals Recombination of Ty elements in yeast can be induced by a double-strand break.

Genetics ◽  
1995 ◽  
Vol 140 (1) ◽  
pp. 67-77 ◽  
Author(s):  
A Parket ◽  
O Inbar ◽  
M Kupiec
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Strand Break ◽  
Double Strand Break ◽  
The Other ◽  
Direct Repeats ◽  
Yeast Saccharomyces Cerevisiae ◽  
Low Frequencies ◽  
Ty Elements ◽  
Main Family

Abstract The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.

scholarly journals The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

2020 ◽  
Vol 44 (3) ◽  
pp. 351-368 ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Christophe Possoz ◽  
David R F Leach
Keyword(s):  
Dna Replication ◽  
Cell Viability ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Initiation ◽  
Genome Replication ◽  
Chromosomal Dna ◽  
Dsb Repair ◽  
Bacterial Dna ◽  
Dna Double Strand Break

ABSTRACT It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

An Immortalised Leukaemia Cell Line Models the Quiescence of Leukaemic Stem Cells and Shows Impaired Double Strand Break Repair Following Daunorubicin Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3989-3989
Author(s):  
Claire Seedhouse ◽  
Abigail Whittall ◽  
Karuna Tandon ◽  
Nigel H. Russell ◽  
Monica Pallis
Keyword(s):  
Cell Cycle ◽  
Strand Break ◽  
Multidrug Resistant ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Proliferating Cells ◽  
Drug Induced ◽  
Strand Breaks ◽  
Damage Response ◽  
Mitochondrial Pore

Abstract Abstract 3989 Objective: Approximately 50% of patients with acute myeloid leukaemia respond to remission-induction chemotherapy, but later relapse. Relapse is thought to be due to the continued presence of a quiescent, chemoresistant leukaemic cell subpopulation. Understanding the damage response in these cells might help to guide targeted therapies. We therefore developed an in vitro model of the quiescent subpopulation and used it to study drug-induced damage and repair in quiescent multidrug resistant cells. Methods: We cultured CD34+ CD38- multidrug resistant KG1a AML cells under several conditions reported to induce cell cycle arrest. We used Pyronin Y to measure RNA content and 7-aminoactinomycin D to measure cell viability. Chemosensitivity, reactive oxygen species (ROS), mitochondrial pore transition and oxidative damage were measured flow cytometrically. gammaH2A.X foci were quantified to measure the double strand break response and DNA damage response and repair gene expression was studied using PCR microarrays and confirmed by real time PCR. Results: mTOR inhibitors induced an increase in G0 without induction of apoptosis. 48 hours' exposure to rapamycin increased the proportion of G0 cells from 13.3% (SD 2.3%) to 46.1% (SD 6%) and decreased mean cell volume. Delayed re-entry into cell cycle following rapamycin withdrawal confirmed the G0 status of these cells. Differentiation markers remained negative. Although several of the other conditions studied resulted in reduced cell growth, they also induced apoptosis, as did combinations of rapamycin with other growth inhibitors. The toxicity of the chemotherapy drug daunorubicin, which acts in part by inducing ROS, was reduced in the quiescence-enriched cells. Sensitivity to mitochondrial pore transition was similar in proliferating and quiescence-enriched cells, indicating that apoptotic pathways are not impaired. However, both basal and drug-induced ROS were significantly lower in quiescence-enriched than in the proliferating cells (p=0.006 for basal ROS and 0.013 for daunorubicin-induced ROS). Furthermore, several DNA repair genes were differentially regulated following daunorubicin treatment of the quiescence-enriched compared to the proliferating cells – these included genes responsible for the repair of double strand breaks. On treatment with daunorubicin, double strand breaks, but not oxidative damage to DNA were observed in both cell populations. However, strikingly, although quiescence-enriched cells sustained fewer DNA damage foci than proliferating cells, they were unable to resolve the damage after daunorubicin was removed. Conclusion: By using rapamycin to enrich KG1a cells for quiescence, we have shown low basal and drug-induced ROS to be associated with chemoresistance in these cells. However, we also found that quiescence gave rise to an impaired double strand break response, which might force these cells to rely on alternative repair pathways and thus be sensitive to synthetic lethal targeting. Disclosures: No relevant conflicts of interest to declare.

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