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Results for “Genome Stability”

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PubMed2002

An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans.

The non-homologous end-joining (NHEJ) pathway of DNA recombination is important for genomic stability in animal cells, since the absence of Ku70, Ku80, Lig4 or Xrcc4 results in non-reciprocal translocation and chromosome fragmentation. The role of LIG4 in the genomic instability of Candida albicans has been analyzed. We have found that both cell transformation and 5'-fluoroorotic acid selection steps used to obtain several lig4 mutants (LIG4/lig4 Ura(+); LIG4/lig4 Ura(-); lig4/lig4 Ura(+); lig4/lig4 Ura(-); and revertant lig4/LIG4 Ura(+)) resulted in significant alterations in chromosome R (ChrR). However, this effect is not specific for LIG4, since disruption of SHE9, a gene unrelated to recombination, also caused alterations in the mobility of ChrR. On the other hand, we could not detect reciprocal or non-reciprocal translocations between non-homologous chromosomes in several lig4 mutants. Furthermore, propagation of these mutants in rich medium did not cause other alterations in the mobility of ChrR. Adaptive mutagenesis of C. albicans, determined by the appearance of L-sorbose-utilizing mutants on L-sorbose plates, was also independent of the presence of Lig4 and occurred by monosomy of Chr5. Accordingly, the NHEJ pathway does not appear to be involved in the adaptive mutagenesis mediated by alterations in chromosome copy number.

Candida albicans↗
PubMed2003

Genomic instability and endoreduplication triggered by RAD17 deletion.

Cell cycle checkpoints are critical for genomic stability. Rad17, a component of the checkpoint clamp loader complex (Rad17/Rfc2-5), is required for the response to DNA damage and replication stress. To explore the role of Rad17 in the maintenance of genomic integrity, we established somatic conditional alleles of RAD17 in human cells. We find that RAD17 is not only important for the Atr-mediated checkpoint but is also essential for cell viability. Cells lacking RAD17 exhibited acute chromosomal aberrations and underwent endoreduplication at a high rate. Therefore, RAD17 links the checkpoint to ploidy control and is essential for the maintenance of chromosomal stability.

Blotting, Southern↗
PubMed2002

V(D)J recombination and DNA repair: lessons from human immune deficiencies and other animal models.

PURPOSE OF REVIEW: V(D)J recombination not only represents the main mechanism for the diversification of the immune system, it also constitutes a critical checkpoint in the development of both B and T lymphocytes. While a defect in V(D)J recombination leads to severe combined immune deficiency, a deregulation of this process can participate in the onset of lymphoid malignancies. RECENT FINDINGS: The careful analysis of human severe combined immune deficiency patients as well as engineered murine models provided several new interesting insights into the physiopathology of the V(D)J recombination process. A new factor of the V(D)J recombination/DNA repair machinery, Artemis, was identified based on its deficiency in human severe combined immune deficiency patients. It also became evident from knockout mouse studies that DNA repair factors that participate in V(D)J recombination can be considered as genomic caretakers. SUMMARY: While V(D)J recombination was first recognized as a critical checkpoint in the development of the immune system, the discovery of several DNA repair factors that participate in this reaction shed light on more general aspects of genomic stability and cancer predisposition.

Animals↗
PubMed2005

The MCT-1 oncogene product impairs cell cycle checkpoint control and transforms human mammary epithelial cells.

Multiple copies in T-cell maligancy (MCT-1) is a putative oncogene initially identified in a human T-cell lymphoma. Forced expression of MCT-1 has recently been shown to induce cell transformation and proliferation, as well as to activate survival-related PI-3K/AKT pathways protecting cells from apoptosis. MCT-1 protein is stabilized in response to DNA damage. The impact of MCT-1 overexpression on DNA damage response remains unknown. Here, we show that MCT-1 deregulates cell cycle checkpoints. The phosphorylation of genomic stabilizers H2AX and NBS1 are enhanced in MCT-1-overexpressing cells. Forced expression of MCT-1 significantly increases the number of DNA damage-induced foci involving gamma-H2AX and 53BP1. In MCT-1-overexpressing cells, the proportion of S-phase cell population is preferentially increased after exposure to gamma-irradiation compared to controls. Knockdown of endogenous MCT-1 using an siRNA approach attenuates the H2AX phosphorylation and the G1/S checkpoint defect. Furthermore, MCT-1 is capable of transforming immortalized human mammary epithelial cells and promoting genomic instability. These data shed light on the role of MCT-1 in the cellular response to DNA damage and its involvement in malignant transformation.

Base Sequence↗
PubMed2003

Aneuploidy and telomere attrition are independent determinants of crisis in SV40-transformed epithelial cells.

Replicative immortality is achieved in vitro by overcoming two mortality checkpoints, M1 (senescence) and M2 (crisis). Cancer cells are thought to overcome M2 by activating telomerase, an enzyme believed to confer genomic stability in addition to maintaining telomeric sequences above a critical length. Here we show that a subset of cultured ovarian cystadenoma cells expressing SV40 large T-antigen, which allows bypassing of M1, develop a specific type of genomic instability, characterized by numerical (as opposed to structural) chromosomal alterations, that leads to non-telomere-based premature growth arrest/crisis. Cells recover from this type of growth arrest and stabilize their ploidy status without telomerase expression. In these cases, telomeres continue to shorten until a second, telomere-based growth arrest/crisis event is reached. Transfection of the catalytic subunit of telomerase does not immortalize cells harboring severe abnormalities in their DNA ploidy but results in immortalization of diploid cells. We conclude that changes in DNA ploidy constitute an important determinant of growth arrest that is independent of telomere attrition in a subset of SV40 large T-antigen-expressing cystadenoma cells. Reestablishment or emergence of ploidy stability, which is not always dependent on telomerase activation, is necessary for acquisition of the potential to achieve replicative immortality.

Aneuploidy↗
PubMed2006

Certainly can't live without this: SIRT6.

Cellular metabolic rates might regulate aging by impinging on genomic stability through the DNA repair pathways. A new study published in Cell (Mostoslavsky et al., 2006) reports that deficiency in one of the mammalian Sir2 homologs, SIRT6, results in genome instability through the DNA base excision repair pathway and leads to aging-associated degenerative phenotypes.

Aging↗
PubMed2000

Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo.

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

Animals↗
PubMed2000

DNA methylation and chromosome instability in lymphoblastoid cell lines.

In order to gain more insight into the relationships between DNA methylation and genome stability, chromosomal and molecular evolutions of four Epstein-Barr virus-transformed human lymphoblastoid cell lines were followed in culture for more than 2 yr. The four cell lines underwent early, strong overall demethylation of the genome. The classical satellite-rich, heterochromatic,juxtacentromeric regions of chromosomes 1, 9, and 16 and the distal part of the long arm of the Y chromosome displayed specific behavior with time in culture. In two cell lines, they underwent a strong demethylation, involving successively chromosomes Y, 9, 16, and 1, whereas in the two other cell lines, they remained heavily methylated. For classical satellite 2-rich heterochromatic regions of chromosomes 1 and 16, a direct relationship could be established between their demethylation, their undercondensation at metaphase, and their involvement in non-clonal rearrangements. Unstable sites distributed along the whole chromosomes were found only when the heterochromatic regions of chromosomes 1 and 16 were unstable. The classical satellite 3-rich heterochromatic region of chromosomes 9 and Y, despite their strong demethylation, remained condensed and stable. Genome demethylation and chromosome instability could not be related to variations in mRNA amounts of the DNA methyltransferases DNMT1, DNMT3A, and DNMT3B and DNA demethylase. These data suggest that the influence of DNA demethylation on chromosome stability is modulated by a sequence-specific chromatin structure.

Ataxia Telangiectasia↗
PubMed2005

A biological network in Saccharomyces cerevisiae prevents the deleterious effects of endogenous oxidative DNA damage.

In this study, we used Saccharomyces cerevisiae to identify a biological network that prevents the deleterious effects of endogenous reactive oxygen species. The absence of Tsa1, a key peroxiredoxin, caused increased rates of mutations, chromosomal rearrangements, and recombination. Defects in recombinational DNA double strand break repair, Rad6-mediated postreplicative repair, and DNA damage and replication checkpoints caused growth defects or lethality in the absence of Tsa1. In addition, the mutator phenotypes caused by a tsa1 mutation were significantly aggravated by defects in Ogg1, mismatch repair, or checkpoints. These results indicate that increased endogenous oxidative stress has broad effects on genome stability and is highly sensitive to the functional state of DNA repair and checkpoints. These findings may provide insight in understanding the consequences of various pathophysiological processes in regard to genomic instability.

DNA Damage↗
PubMed1998

Yeast ARMs (DNA at-risk motifs) can reveal sources of genome instability.

The genomes of all organisms contain an abundance of DNA repeats which are at-risk for causing genetic change. We have used the yeast Saccharomyces cerevisiae to investigate various repeat categories in order to understand their potential for causing genomic instability and the role of DNA metabolism factors. Several types of repeats can increase enormously the likelihood of genetic changes such as mutation or recombination when present either in wild type or mutants defective in replication or repair. Specifically, we have investigated inverted repeats, homonucleotide runs, and short distant repeats and the consequences of various DNA metabolism mutants. Because the at-risk motifs (ARMs) that we characterized are sensitive indicators, we have found that they are useful tools to reveal new genetic factors affecting genome stability as well as to distinguish subtle differences between alleles.

Animals↗
PubMed2006

Dynamic roles for G4 DNA in the biology of eukaryotic cells.

Recent advances have made a persuasive case for the existence of G4 DNA in living cells, but what--if any--are its functions? Experiments have established how G4 DNA may contribute to the biology of eukaryotic cells, and genomic analysis has identified new ways in which the potential to form G4 DNA may influence gene regulation and genomic stability. This Perspective highlights those advances and identifies some key open questions.

Animals↗
PubMed2025

Decoding nucleoside supplementation: how thymidine outperforms ribonucleosides in accelerating mammalian replication forks.

Disruptions in deoxynucleoside triphosphate (dNTP) supply impair DNA replication and lead to genomic instability. While exogenous ribonucleosides (rNuc) have been suggested to alleviate replication stress by increasing dNTP levels, their precise metabolic effects remain unclear. Here, we show that rNuc supplementation primarily elevates CTP and UTP levels, with only modest increases in dCTP, and has minimal impact on replication fork speed across multiple mammalian cell lines. In contrast, thymidine (dThd), either alone or in combination with rNuc-as in EmbryoMax Nucleosides-significantly increases dTTP and dGTP levels, leading to accelerated replication fork progression. Notably, dThd, rather than rNuc, drives fork acceleration and counteracts fork slowdown caused by elevated dUTP, consistent with primer extension assays showing that dUTP transiently inhibits Pol ϵ-mediated DNA synthesis at template adenines. These results clarify the distinct roles of nucleosides in nucleotide metabolism, providing a mechanistic basis for how dThd promotes fork progression and preserves genomic stability.

DNA Replication↗
PubMed2006

Replication-associated gene dosage effects shape the genomes of fast-growing bacteria but only for transcription and translation genes.

The bidirectional replication of bacterial genomes leads to transient gene dosage effects. Here, we show that such effects shape the chromosome organisation of fast-growing bacteria and that they correlate strongly with maximal growth rate. Surprisingly the predicted maximal number of replication rounds shows little if any phylogenetic inertia, suggesting that it is a very labile trait. Yet, a combination of theoretical and statistical analyses predicts that dozens of replication forks may be simultaneously present in the cells of certain species. This suggests a strikingly efficient management of the replication apparatus, of replication fork arrests and of chromosome segregation in such cells. Gene dosage effects strongly constrain the position of genes involved in translation and transcription, but not other highly expressed genes. The relative proximity of the former genes to the origin of replication follows the regulatory dependencies observed under exponential growth, as the bias is stronger for RNA polymerase, then rDNA, then ribosomal proteins and tDNA. Within tDNAs we find that only the positions of the previously proposed 'ubiquitous' tRNA, which translate the most frequent codons in highly expressed genes, show strong signs of selection for gene dosage effects. Finally, we provide evidence for selection acting upon genome organisation to take advantage of gene dosage effects by identifying a positive correlation between genome stability and the number of simultaneous replication rounds. We also show that gene dosage effects can explain the over-representation of highly expressed genes in the largest replichore of genomes containing more than one chromosome. Together, these results demonstrate that replication-associated gene dosage is an important determinant of chromosome organisation and dynamics, especially among fast-growing bacteria.

Bacteria↗
PubMed2006

Evaluating the delayed effects of cellular exposure to ionizing radiation.

A number of ongoing delayed effects have now been described in the progeny of an irradiated cell. These are grouped under the rubric of radiation induced genomic instability. Perhaps the best characterized is the dynamic production of chromosomal rearrangements in some clonally expanded cells surviving irradiation. In this chapter we provide the protocols for irradiation, cell culture, chromosome analysis, and characterization of the status of genomic stability in the context of delayed radiation effects.

Cells, Cultured↗
PubMed2006

Substrates of the BRCA1-dependent ubiquitin ligase.

Discovering the precise function of the breast and ovarian specific tumor suppressor, BRCA1, has proven to be quite complicated. It has been determined that BRCA1, together with BARD1, comprise an E3 ubiquitin ligase. Since it is now known that BRCA1 is an enzyme, the challenge for BRCA1 research is to learn how this enzymatic activity functions in normal breast and ovarian cells in order to suppress cancerous transformation. This review will survey the known ubiquitination substrates of BRCA1 and suggest how these reactions may influence the genomic stability and proliferation of breast cells.

BRCA1 Protein↗
PubMed2005

Comparative genomic hybridization profiles in human BRCA1 and BRCA2 breast tumors highlight differential sets of genomic aberrations.

BRCA1 or BRCA2 germline mutations cause approximately 30% of breast cancers within high-risk families. This represents 5% of total breast cancer incidence. Although BRCA1 and BRCA2 are both implicated in DNA repair and genome stability, it is unknown whether BRCA1 and BRCA2 are associated with similar or distinct diseases. In a previous study we reported that BRCA1-related breast carcinomas show a distinct genomic profile as determined by comparative genomic hybridization (CGH). We now hypothesize that, if functionally equivalent, mutations in BRCA1 and BRCA2 would result in similar genomic profiles in tumors. Here we report the chromosomal gains and losses as measured by CGH in 25 BRCA2-associated breast tumors and compared them with our existing 36 BRCA1 and 30 control profiles. We compared all chromosomal regions and determined the regions of differential gain or loss between tumor classes and controls. BRCA2 and control tumors have very similar genomic profiles. As a consequence, and in contrast to BRCA1-associated tumors, CGH profiles from BRCA2-associated tumors could not be distinguished from control tumors using the classification methodology as we have developed before. The largest number of significant differences existed between BRCA1 and controls, followed by BRCA1 compared with BRCA2, suggesting different tumor development pathways for BRCA1 and BRCA2.

Breast Neoplasms↗
PubMed2004

SUMO: a regulator of gene expression and genome integrity.

Post-translational modification with the ubiquitin-like SUMO protein is involved in the regulation of many cellular key processes. The SUMO system modulates signal transduction pathways, including cytokine, Wnt, growth factor and steroid hormone signalling. SUMO frequently restrains the activity of downstream transcription factors in these pathways presumably by facilitating the recruitment of corepressors or mediating the assembly of repressor complexes. Additionally, evidence is accumulating that SUMO controls pathways important for the surveillance of genome integrity. SUMO regulates the PML/p53 tumour suppressor network, a key determinant in the cellular response to DNA damage. Moreover, proteins that maintain genomic stability by functioning at the interface between DNA replication, recombination and repair processes undergo SUMOylation. We will discuss some key findings that exemplify the role of SUMO in transcriptional regulation and genome surveillance.

Animals↗
PubMed2004

A role for the Fanconi anemia C protein in maintaining the DNA damage-induced G2 checkpoint.

Fanconi anemia (FA) is a complex, heterogeneous genetic disorder composed of at least 11 complementation groups. The FA proteins have recently been found to functionally interact with the cell cycle regulatory proteins ATM and BRCA1; however, the function of the FA proteins in cell cycle control remains incompletely understood. Here we show that the Fanconi anemia complementation group C protein (Fancc) is necessary for proper function of the DNA damage-induced G2/M checkpoint in vitro and in vivo. Despite apparently normal induction of the G2/M checkpoint after ionizing radiation, murine and human cells lacking functional FANCC did not maintain the G2 checkpoint as compared with wild-type cells. The increased rate of mitotic entry seen in Fancc-/-mouse embryo fibroblasts correlated with decreased inhibitory phosphorylation of cdc2 kinase on tyrosine 15. An increased inability to maintain the DNA damage-induced G2 checkpoint was observed in Fancc -/-; Trp53 -/-cells compared with Fancc -/-cells, indicating that Fancc and p53 cooperated to maintain the G2 checkpoint. In contrast, genetic disruption of both Fancc and Atm did not cooperate in the G2 checkpoint. These data indicate that Fancc and p53 in separate pathways converge to regulate the G2 checkpoint. Finally, fibroblasts lacking FANCD2 were found to have a G2 checkpoint phenotype similar to FANCC-deficient cells, indicating that FANCD2, which is activated by the FA complex, was also required to maintain the G2 checkpoint. Because a proper checkpoint function is critical for the maintenance of genomic stability and is intricately related to the function and integrity of the DNA repair process, these data have implications in understanding both the function of FA proteins and the mechanism of genomic instability in FA.

Alleles↗