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At least 343 records · Page 19Linked to original sources
PubMed2002

BRCA1 facilitates microhomology-mediated end joining of DNA double strand breaks.

BRCA1 is critical for the maintenance of genomic stability, in part through its interaction with the Rad50.Mre11.Nbs1 complex, which occupies a central role in DNA double strand break repair mediated by nonhomologous end joining (NHEJ) and homologous recombination. BRCA1 has been shown to be required for homology-directed recombination repair. However, the role of BRCA1 in NHEJ, a critical pathway for the repair of double strand breaks and genome stability in mammalian cells, remains elusive. Here, we established a pair of mouse embryonic fibroblasts (MEFs) derived from 9.5-day-old embryos with genotypes Brca1(+/+):p53(-/-) or Brca1(-/-):p53(-/-). The Brca1(-/-):p53(-/-) MEFs appear to be extremely sensitive to ionizing radiation. The contribution of BRCA1 in NHEJ was evaluated in these cells using three different assay systems. First, transfection of a linearized plasmid in which expression of the reporter gene required precise end joining indicated that Brca1(-/-) MEFs display a moderate deficiency when compared with Brca1(+/+) cells. Second, using a retrovirus infection assay dependent on NHEJ, a 5-10-fold reduction in retroviral integration efficiency was observed in Brca1(-/-) MEFs when compared with the Brca1(+/+) MEFs. Third, Brca1(-/-) MEFs exhibited a 50-100-fold deficiency in microhomology-mediated end-joining activity of a defined chromosomal DNA double strand break introduced by a rare cutting endonuclease I-SceI. These results provide evidence that Brca1 has an essential role in microhomology-mediated end joining and suggest a novel molecular basis for its caretaker role in the maintenance of genome integrity.

Adenoviridae↗
PubMed2005

53BP1 cooperates with p53 and functions as a haploinsufficient tumor suppressor in mice.

p53 binding protein 1 (53BP1) is a putative DNA damage sensor that accumulates at sites of double-strand breaks (DSBs) in a manner dependent on histone H2AX. Here we show that the loss of one or both copies of 53BP1 greatly accelerates lymphomagenesis in a p53-null background, suggesting that 53BP1 and p53 cooperate in tumor suppression. A subset of 53BP1-/- p53-/- lymphomas, like those in H2AX-/- p53-/- mice, were diploid and harbored clonal translocations involving antigen receptor loci, indicating misrepair of DSBs during V(D)J recombination as one cause of oncogenic transformation. Loss of a single 53BP1 allele compromised genomic stability and DSB repair, which could explain the susceptibility of 53BP1+/- mice to tumorigenesis. In addition to structural aberrations, there were high rates of chromosomal missegregation and accumulation of aneuploid cells in 53BP1-/- p53+/+ and 53BP1-/- p53-/- tumors as well as in primary 53BP1-/- splenocytes. We conclude that 53BP1 functions as a dosage-dependent caretaker that promotes genomic stability by a mechanism that preserves chromosome structure and number.

Alleles↗
PubMed2003

Mammalian longevity under the protection of PARP-1's multi-facets.

Given the presence of continuous endogenous and exogenous sources of stress, mammalian species have evolved complex systems of protection, detoxification and repair, in order to maintain homeostasis during development and until reproductive maturity for the sake of the species. However, since no system is perfect, complete prevention of damage is unlikely to occur. Accumulation of macromolecular damage, including damage to DNA and genomic instability, is considered a driving force for the ageing process and age-related diseases. One of the immediate eukaryotic cellular responses to DNA breakage is the covalent post-translational modification of nuclear proteins with poly(ADP-ribose) from NAD+ as precursor, mostly catalysed by poly(ADP-ribose) polymerase-1 (PARP-1). Poly(ADP-ribosyl)ation is involved in DNA base-excision repair (BER), DNA-damage signalling and regulation of genomic stability. In recent years, many groups have become involved in PARP field, shedding light on new partners for PARP-1, new members of the PARP family and new physiological and pathophysiological properties for the founding member of the poly(ADP-ribose) polymerase super family. The present review focuses on PARP-1 and its role in the maintenance of genome stability and in mammalian longevity.

Aging↗
PubMed2000

DNA stability and genomic methylation status in colonocytes isolated from methyl-donor-deficient rats.

BACKGROUND: Epidemiological studies report an inverse relationship between intake of the B vitamin folic acid and colon cancer. Folate is important for DNA synthesis and repair. Moreover, the production of S-adenosylmethionine (SAM), essential for normal DNA methylation and gene expression, is dependent on folic acid. Folate deficiency may increase the risk of malignant transformation by perturbing these pathways. AIMS OF THE STUDY: The principal aim of this study was to determine the effects of folate deficiency on DNA stability and DNA methylation in rat colonocytes in vivo. As the metabolic pathways of folate and other dietary methyl donors are closely linked, the effects of methionine and choline deficiency were also evaluated. METHODS: Male Hooded-Lister rats were fed a diet deficient in folic acid, or in methionine and choline, or in folate, methionine and choline for 10 weeks. DNA strand breakage and misincorporated uracil were determined in isolated colonocytes using alkaline single cell gel electrophoresis. Global DNA methylation was measured in colonic scrapings. Folate was measured in plasma, erythrocyte and liver samples. RESULTS: Methyl donor deficiency induced DNA strand breakage in colonocytes isolated from all experimental groups. Uracil levels in colonocyte DNA remained unchanged compared with controls. DNA methylation was unaffected either by folate and/or methionine and choline depletion. Rats fed a folate-deficient diet had less folate in plasma, red blood cells and liver than controls. CONCLUSIONS: Folate and methyl deficiency in vivo primarily affects DNA stability in isolated colonocytes of rats, without affecting overall DNA methylation.

Animals↗
PubMed2025

The dynamic centromere.

Centromeres are fundamental chromosomal structures that ensure accurate chromosome segregation during cell division. Despite their conserved and essential role in maintaining genomic stability, centromeres are subject to rapid evolutionary change. At the heart of centromere identity is the histone H3 variant CENP-A, an epigenetic mark that defines and propagates active centromeres and is essential for their function. Recent evidence supports a rapid evolution of centromere DNA sequences but also suggests a certain degree of flexibility in CENP-A deposition and propagation. The phenomenon of centromere drift, recently observed in humans, highlights how the dynamic repositioning of CENP-A and associated epigenetic environment over time maintains a regulated equilibrium, ensuring centromere function despite positional variation. Understanding these processes is crucial for unraveling centromere dynamics and their broader implications for genome stability and evolution.

Centromere↗
PubMed2006

The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants.

DNA replication in cycling eukaryotic cells necessitates the reestablishment of chromatin after nucleosome redistribution from the parental to the two daughter DNA strands. Chromatin assembly factor 1 (CAF-1), a heterotrimeric complex consisting of three subunits (p150/p60/p48), is one of the replication-coupled assembly factors involved in the reconstitution of S-phase chromatin. CAF-1 is required in vitro for nucleosome assembly onto newly replicated chromatin in human cells and Arabidopsis thaliana, and defects in yeast (Saccharomyces cerevisiae) affect DNA damage repair processes, predominantly those involved in genome stability. However, in vivo chromatin defects of caf-1 mutants in higher eukaryotes are poorly characterized. Here, we show that fasciata1-4 (fas1-4), a new allele of the Arabidopsis fas1 mutant defective in the p150 subunit of CAF-1, has a severe developmental phenotype, reduced heterochromatin content, and a more open conformation of euchromatin. Most importantly, homologous recombination (HR), a process involved in maintaining genome stability, is increased dramatically in fas1-4, as indicated by a 96-fold stimulation of intrachromosomal HR. Together with the open conformation of chromatin and the nearly normal expression levels of HR genes in the mutant, this result suggests that chromatin is a major factor restricting HR in plants.

Arabidopsis↗
PubMed2003

p55CDC/hCDC20 mutant induces mitotic catastrophe by inhibiting the MAD2-dependent spindle checkpoint activity in tumor cells.

Nondisjunction of chromosomes results in aneuploidy in mammalian cells causing genomic instability. The spindle checkpoint, one of the surveillance systems to maintain genomic stability, prevents missegregation of chromosomes until all the kinetochores are properly attached with bipolar spindles. When this condition is not met, MAD2, a component of the spindle checkpoint complex, associates with p55CDC/hCDC20 to inhibit ubiquitination of substrates by the anaphase-promoting complex (APC). In this study, we have focused on the biological role of the MAD2-binding domain in p55CDC/hCDC20 in the maintenance of genomic stability. Based on previous studies, we constructed a truncated p55CDC/hCDC20 mutant (F2) that harbors only the MAD2-binding domain. Interestingly, we found that in the yeast two-hybrid system, the interaction of F2 and MAD2 was stronger than that of intact p55CDC/hCDC20. We also found that in the presence of the microtubule-disrupting drug, nocodazole, U2OS cells expressing p55CDC/hCDC20 mutants bypassed the mitotic arrest and showed apoptotic morphologies, whereas cells harboring vector alone arrested at metaphase. In particular, the apoptotic phenomena were dramatically enhanced in the F2-expressing cells. These mitotic catastrophes also occurred in cells treated with other microtubule disrupting agents, such as taxol and vinblastine. In addition, the mutant cells exhibited chromosomal missegregation during mitosis, even in the absence of nocodazole. Taken together, these results suggest that agents blocking the spindle checkpoint response may induce tumor cells to become more sensitive to spindle poison drugs, providing a powerful tool to improve chemotherapy.

Antineoplastic Agents↗
PubMed2003

Pathogen-induced systemic plant signal triggers DNA rearrangements.

Plant genome stability is known to be affected by various abiotic environmental conditions, but little is known about the effect of pathogens. For example, exposure of maize plants to barley stripe mosaic virus seems to activate transposable elements and to cause mutations in the non-infected progeny of infected plants. The induction by barley stripe mosaic virus of an inherited effect may mean that the virus has a non-cell-autonomous influence on genome stability. Infection with Peronospora parasitica results in an increase in the frequency of somatic recombination in Arabidopsis thaliana; however, it is unclear whether effects on recombination require the presence of the pathogen or represent a systemic plant response. It is also not clear whether the changes in the frequency of somatic recombination can be inherited. Here we report a threefold increase in homologous recombination frequency in both infected and non-infected tissue of tobacco plants infected with either tobacco mosaic virus or oilseed rape mosaic virus. These results indicate the existence of a systemic recombination signal that also results in an increased frequency of meiotic and/or inherited late somatic recombination.

Gene Expression Regulation, Plant↗
PubMed2006

Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes.

Folic acid plays a key role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for DNA methylation. Besides dietary habits, folic acid metabolism is influenced by genetic polymorphism. The C677T polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene is associated with a reduction of catalytic activity and is suggested to modify cancer risk differently depending on folate status. In this work the effect of folic acid deficiency on genome stability and radiosensitivity has been investigated in cultured lymphocytes of 12 subjects with different MTHFR genotype (four for each genotype). Cells were grown for 9 days with 12, 24 and 120 nM folic acid and analyzed in a comprehensive micronucleus test coupled with centromere characterization by CREST immunostaining. In other experiments, cells were grown with various folic acid concentrations, irradiated with 0.5 Gy of gamma rays and analyzed in the micronucleus test. The results obtained indicate that folic acid deficiency induces to a comparable extent chromosome loss and breakage, irrespective of the MTHFR genotype. The effect of folic acid was highly significant (P < 0.001) and explained >50% of variance of both types of micronuclei. Also nucleoplasmic bridges and buds were significantly increased under low folate supply; the increase in bridges was mainly observed in TT cells, highlighting a significant effect of the MTHFR genotype (P = 0.006) on this biomarker. Folic acid concentration significantly affected radiation-induced micronuclei (P < 0.001): the increased incidence of radiation-induced micronuclei with low folic acid was mainly accounted for by carriers of the variant MTHFR allele (both homozygotes and heterozygotes), but the overall effect of genotype did not attain statistical significance. Treatment with ionizing radiations also increased the frequency of nucleoplasmic bridges. The effect of folic acid level on this end-point was modulated by the MTHFR genotype (P for interaction = 0.02), with TT cells grown at low folic acid concentration apparently resistant to the induction of radiation-induced bridges. Finally, the effect of in vitro folate deprivation on global DNA methylation was evaluated in lymphocytes of six homozygous subjects (three CC and three TT). The results obtained suggest that, under the conditions of this work, folic acid deprivation is associated with global DNA hypermethylation.

Adult↗
PubMed1997

When helicase and topoisomerase meet!

Several examples of direct interactions between helicases and topoisomerases have recently been described. The data suggest a possible cooperation between these enzymes in major DNA events such as the progression of a replication fork, segregation of newly replicated chromosomes, disruption of nucleosomal structure, DNA supercoiling, and finally recombination, repair, and genomic stability. A first example is the finding of a strong interaction between T antigen and topoisomerase I in mammalian cells, that may trigger unwinding of the parental DNA strands at the replication forks of Simian Virus 40. A second example is the reverse gyrase from thermophilic prokaryotes, composed of a putative helicase domain, and a topoisomerase domain in the same polypeptide. This enzyme may be required to maintain genomic stability at high temperature. A third example is the finding of an interaction between type II topoisomerase and the helicase Sgs1 in yeast. This interaction possibly allows the faithful segregation of newly replicated chromosomes in eukaryotic cells. A fourth example is the interaction between the same helicase Sgs1 and topoisomerase III in yeast, that may control recombination level and genetic stability of repetitive sequences. Recently, in humans, mutations in genes similar to Sgs1 have been found to be responsible for Bloom's and Werner's syndromes. The cooperation between helicases and topoisomerases is likely to be extended to many aspects of DNA mechanisms including chromatin condensation/decondensation.

Archaea↗
PubMed2002

Genetic and epigenetic alterations in colon cancer.

Colorectal cancer affected approximately 135,000 people in the United States in 2001, resulting in 57,000 deaths. Colorectal cancer develops as the result of the progressive accumulation of genetic and epigenetic alterations that lead to the transformation of normal colonic epithelium to colon adenocarcinoma. The loss of genomic stability is a key molecular and pathophysiologic step in this process and serves to create a permissive environment for the occurrence of alterations in tumor suppressor genes and oncogenes. Alterations in these genes, which include APC, CTNNB1, K-RAS, MADH4/SMAD4, and TGFBR2, appear to promote colon tumorigenesis by perturbing the function of signaling pathways, such as the TGF-ss signaling pathway, or by affecting genes that regulate genomic stability, such as the mutation mismatch repair genes.

Adenocarcinoma↗
PubMed2005

BRCA1 participates in DNA decatenation.

The tumor suppressor BRCA1 has an important function in the maintenance of genomic stability. Increasing evidence suggests that BRCA1 regulates cell cycle checkpoints and DNA repair after DNA damage. However, little is known about its normal function in the absence of DNA damage. Here we show that BRCA1 interacts and colocalizes with topoisomerase IIalpha in S phase cells. Similar to cells treated with the topoisomerase IIalpha inhibitor ICRF-193, BRCA1-deficient cells show lagging chromosomes, indicating a defect in DNA decatenation and chromosome segregation. More directly, BRCA1 deficiency results in defective DNA decatenation in vitro. Finally, topoisomerase IIalpha is ubiquitinated in a BRCA1-dependent manner, and topoisomerase IIalpha ubiquitination correlates with higher DNA decatenation activity. Together these results suggest an important role of BRCA1 in DNA decatenation and reveal a previously unknown function of BRCA1 in the maintenance of genomic stability.

Antigens, Neoplasm↗
PubMed2001

P53-responsive genes and the potential for cancer diagnostics and therapeutics development.

P53 protein regulates cell responses to DNA damage to keep genomic stability by transactivation and trans-repression of its downstream target genes. P53 protein also has activators, inactivators, or co-factors via interaction with other proteins. Both the p53-regulated genes and interacted proteins form a huge network. As tumors usually escape from proliferating controls by means of accumulation of genetic alterations, p53 is one of the most important tumor suppressor genes that can be targeted for diagnosis, prognosis, and therapeutic intervention. Reviewing the p53-network is of great importance. In this review, we are focusing on cancer-related p53 downstream-regulated genes. Various methods dealing with the discovery of p53-regulated genes by the detection of gene expression have been applied. Recently high throughput functional genomics methods, such as DNA microarray, serial analysis of gene expression (SAGE), differential display, and protein two-dimensional gel electrophoresis, have provided a wealth of information on the dynamics of cell context responses. Hundreds of genes have been discovered whose transcriptions are regulated by p53 protein. They were grouped, based on their functions, into sub-classes including cell-cycle regulation, DNA repair, angiogenesis, metastasis, and multidrug resistance. P53 plays a pivotal role in keeping genomic stability and tumor suppression. The deeper we investigate the cell responses as mediated by p53, the more complex p53-network becomes. However, understanding p53-network, offers great opportunities to develop more sensitive and accurate diagnostic/prognostic tools, as well as more efficient therapies for cancer.

Apoptosis↗
PubMed2005

[Direct role of p53 on homologous recombination].

The tumor suppressor gene p53, which is the most frequently mutated gene in human tumors, controls cell cycle checkpoint and apoptosis via the transactivation of the transcription of a collection of genes. These activities avoid proliferation of cell bearing alteration of genetic material. However, like a two-edged sword, p53 can also directly participate to genome stability maintenance by repressing homologous recombination (HR), independently of the transactivation activity. This parallel activity allows to limit the deleterious consequences on an excess of HR. Beside genetic interactions, p53 protein physically interacts with both HR proteins and HR intermediates (heteroduplex and Holliday junctions). The core domain of p53 is required for interaction with Rad51 at an early step and the carboxy-terminal domain of p53 is involved in the interaction with Rad54 and HR intermediates, at a late step. We discuss here the putative consequences of this parallel activity of p53 on genome stability, speciation and tumor protection.

DNA-Binding Proteins↗
PubMed2007

BRCA1 contributes to cell cycle arrest and chemoresistance in response to the anticancer agent irofulven.

Tumor suppressor gene BRCA1 is frequently mutated in familial breast and ovarian cancer. BRCA1 plays pivotal roles in maintaining genomic stability by interacting with numerous proteins in cell cycle control and DNA repair. Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is one of a new class of anticancer agents that are analogs of mushroom-derived illudin toxins. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor cell types. The exact nature of irofulven-induced DNA damage is not completely understood. We demonstrated previously that irofulven activates ATM and its targets, NBS1, SMC1, CHK2, and p53. In this study, we hypothesize that irofulven induces DNA double-strand breaks and that BRCA1 may affect chemosensitivity by controlling cell cycle checkpoints, DNA repair, and genomic stability in response to irofulven treatment. We observed that irofulven induces the formation of chromosome breaks and radials and the activation and foci formation of gamma-H2AX, BRCA1, and RAD51. We also provided evidence that irofulven induces the generation of DNA double-strand breaks. By using BRCA1-deficient or -proficient cells, we demonstrated that in response to irofulven, BRCA1 contributes to the control of S and G(2)/M cell cycle arrest and is critical for repairing DNA double-strand breaks and for RAD51-dependent homologous recombination. Furthermore, we found that BRCA1 deficiency results in increased chromosome damage and chemosensitivity after irofulven treatment.

Antineoplastic Agents↗
PubMed2005

Current advances in unraveling the function of the Werner syndrome protein.

Werner syndrome (WS) is an autosomal recessive premature aging disease manifested by the mimicry of age-related phenotypes such as atherosclerosis, arteriosclerosis, cataracts, osteoporosis, soft tissue calcification, premature thinning, graying, and loss of hair, as well as a high incidence of some types of cancers. The gene product defective in WS, WRN, is a member of the RecQ family of DNA helicases that are widely distributed in nature and believed to play central roles in genomic stability of organisms ranging from prokaryotes to mammals. Interestingly, WRN is a bifunctional protein that is exceptional among RecQ helicases in that it also harbors an exonuclease activity. Furthermore, it preferentially operates on aberrant DNA structures believed to exist in vivo as intermediates in specific DNA transactions such as replication (forked DNA), recombination (Holliday junction, triplex and tetraplex DNA), and repair (partial duplex with single stranded bubble). In addition, WRN has been shown to physically and functionally interact with a variety of DNA-processing proteins, including those that are involved in resolving alternative DNA structures, repair DNA damage, and provide checkpoints for genomic stability. Despite significant research activity and considerable progress in understanding the biochemical and molecular genetic function of WRN, the in vivo molecular pathway(s) of WRN remain elusive. The following review focuses on the recent advances in the biochemistry of WRN and considers the putative in vivo functions of WRN in light of its many protein partners.

DNA Helicases↗
PubMedDate not supplied

Role of p53 and apoptosis in carcinogenesis.

Carcinogenesis is a process that converts normal cells from controllable to uncontrollable growth. Coordinate regulation of the rates of cell proliferation and cell death is an important determinant in maintenance of homeostasis. Loss of control of this balance is central to the development of cancer. This loss may be due to genetic alteration in either growth promoting genes resulting in constitutive activation or negative growth regulating genes such as tumor suppressor genes. Recent advances in studying the molecular mechanisms related to the etiology of cancer have provided further understanding of these pathways. Earlier studies have been primarily concerned with cell proliferation resulting from activation of oncogenes. However, many recent studies have focused on the induction of cell death. The recognition of the importance of apoptosis, a distinct mode of cell death, in maintenance of genomic stability was further prompted by studying the mechanism of the tumor suppressor gene product p53, as well as many other oncogenes and tumor suppressor genes. For example, many oncogenes appear to act as potent inducers of apoptosis through activation of p53 dependent apoptosis pathways. Therefore, one possible mechanism for tumor suppression involves activation of apoptosis pathways in cells at risk of neoplastic transformation. These studies have provided extensive knowledge of the signal transduction pathways in response to genotoxic stress and promoted mechanistic research related to the apoptosis pathways. These studies also provide a perfect explanation that p53 is a key element in maintaining genomic stability and loss of the p53 function is a common event during carcinogenesis. This chapter will mainly focus on the role of apoptosis in carcinogenesis. In particular, I will summarize recent studies related to the mechanisms of p53 and its role in this process.

Apoptosis↗
PubMed1999

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53 -/- normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53 +/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53-/- normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.

Animals↗