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Results for “Homologous Recombination Deficiency”

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At least 127 records · Page 7Linked to original sources

PubMed1997

Impaired diurnal adrenal rhythmicity restored by constant infusion of corticotropin-releasing hormone in corticotropin-releasing hormone-deficient mice.

The normal pattern of daily glucocorticoid production in mammals requires circadian modulation of hypothalamicpituitary-adrenal axis activity. To assess both the factors responsible for imparting this diurnal profile and its physiologic importance, we have exploited corticotropin-releasing hormone (CRH)-deficient mice generated by homologous recombination in embryonic stem cells. CRH-deficient mice have lost normal circadian variations in plasma ACTH and glucocorticoid while maintaining normal circadian locomotor activity. Constant peripheral infusion of CRH produced marked diurnal excursions of plasma glucocorticoid, indicating that CRH acts in part as a permissive factor for other circadian modulators of adrenocortical activity. The presence of atrophic adrenals in CRH-deficient mice without an overt deficit in basal plasma ACTH concentration suggests that the diurnal increase in ACTH is essential to maintain normal adrenal function.

Adrenal Cortex↗

PubMed2005

Role of the Rep helicase gene in homologous recombination in Neisseria gonorrhoeae.

In Escherichia coli, the Rep helicase has been implicated in replication fork progression, replication restart, homologous recombination, and DNA repair. We show that a Neisseria gonorrhoeae rep mutant is deficient in the homologous-recombination-mediated processes of DNA transformation and pilus-based colony variation but not in DNA repair.

Amino Acid Sequence↗

PubMed1990

Relative frequencies of homologous recombination between plasmids introduced into DNA repair-deficient and other mammalian somatic cell lines.

Twelve mammalian somatic cell lines, some of them DNA damage-sensitive mutants paired with their respective wild-type parental lines, were assayed for their ability to catalyze extrachromosomal, intermolecular homologous recombination between pSV2neo plasmid recombination substrates. All of the somatic cell lines analyzed are capable of catalyzing homologous recombination; however, there is a wide range of efficiencies with which they do so. Five human cell lines display a fourfold range of recombination frequencies, and six hamster cell lines vary almost 20-fold. Linearizing one of the recombination substrates stimulates recombination in all but one of the cell lines. Two of the three paired mutant cell lines display a threefold reduction in their ability to catalyze homologous recombination when compared to their respective parental cell lines, indicating that the mutations that render them sensitive to DNA damaging agents might also play a role in homologous recombination.

Agammaglobulinemia↗

PubMed2004

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay.

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.

Animals↗

PubMed2005

Hypersensitivity of Brca1-deficient MEF to the DNA interstrand crosslinking agent mitomycin C is associated with defect in homologous recombination repair and aberrant S-phase arrest.

Hypersensitivity of Brca1-deficient cells to interstrand crosslinking (ICL) agents such as cisplatin and mitomycin C (MMC) implicates an important role for Brca1 in cellular response to the ICL DNA damage repair. However, the detailed mechanism of how Brca1 is involved in the ICL response remains unclear. In this study, we analysed the cellular response to MMC treatment using isogenic mouse embryonic fibroblasts (MEFs) including wild type, p53-/- and p53-/-Brca1-/-. Marked hypersensitivity of p53-/- Brca1-/- MEFs to MMC was found, and the reconstitution of Brca1 expression in these cells restored resistance to MMC. Upon MMC treatment, wild-type MEF was temporarily arrested at G2/M phase but subsequently resumed a normal cell cycle progression. In contrast, Brca1-deficient MEF exhibited a marked time-dependent accumulation of cells arrested at S phase and a prolonged increase in the G2/M population, followed by extensive cell deaths. Importantly, DNA damage-induced Rad51 foci were not formed in these cells, suggesting a defect in homologous recombination. Such defects are fully rescued by reconstitution of Brca1 expression in Brca1-deficient MEF, suggesting that Brca1 directly plays an essential role in ICL repair, which depends on homologous recombination during S phase.

Animals↗

PubMed2002

Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants.

The Saccharomyces cerevisiae TOP3 gene encodes the type IA topoisomerase (Top3p) that is highly conserved in evolution. Deletion of TOP3 leads to a reduction in cell viability, hyper-recombination between repetitive DNA sequences, and abnormalities in both cell cycle progression and responses to DNA damaging agents. Deletion of SGS1, encoding the sole RecQ family helicase in S. cerevisiae, strongly suppresses the phenotypic effects of loss of TOP3 function. Here, we show that many of the adverse phenotypic effects of TOP3 deletion can also be partially alleviated by disruption of homologous recombination (HR) functions. This genetic interaction is seen both in strains deleted for TOP3 and in wild-type strains over-expressing a dominant-negative Top3p mutant form that confers a top3-like phenotype. Moreover, we show that this genetic interaction is conserved in the distantly-related fission yeast, Schizosaccharomyces pombe. Our results implicate topoisomerase III enzymes in recombination repair events required for cellular protection against DNA damaging agents and DNA replication inhibitors.

Blotting, Western↗

PubMed2000

Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo.

Multiple inositol polyphosphate phosphatase (Minpp1) metabolizes inositol 1,3,4,5,6-pentakisphosphate (InsP(5)) and inositol hexakisphosphate (InsP(6)) with high affinity in vitro. However, Minpp1 is compartmentalized in the endoplasmic reticulum (ER) lumen, where access of enzyme to these predominantly cytosolic substrates in vivo has not previously been demonstrated. To gain insight into the physiological activity of Minpp1, Minpp1-deficient mice were generated by homologous recombination. Tissue extracts from Minpp1-deficient mice lacked detectable Minpp1 mRNA expression and Minpp1 enzyme activity. Unexpectedly, Minpp1-deficient mice were viable, fertile, and without obvious defects. Although Minpp1 expression is upregulated during chondrocyte hypertrophy, normal chondrocyte differentiation and bone development were observed in Minpp1-deficient mice. Biochemical analyses demonstrate that InsP(5) and InsP(6) are in vivo substrates for ER-based Minpp1, as levels of these polyphosphates in Minpp1-deficient embryonic fibroblasts were 30 to 45% higher than in wild-type cells. This increase was reversed by reintroducing exogenous Minpp1 into the ER. Thus, ER-based Minpp1 plays a significant role in the maintenance of steady-state levels of InsP(5) and InsP(6). These polyphosphates could be reduced below their natural levels by aberrant expression in the cytosol of a truncated Minpp1 lacking its ER-targeting N terminus. This was accompanied by slowed cellular proliferation, indicating that maintenance of cellular InsP(5) and InsP(6) is essential to normal cell growth. Yet, depletion of cellular inositol polyphosphates during erythropoiesis emerges as an additional physiological activity of Minpp1; loss of this enzyme activity in erythrocytes from Minpp1-deficient mice was accompanied by upregulation of a novel, substitutive inositol polyphosphate phosphatase.

3T3 Cells↗

PubMed2006

Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines.

The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.

Animals↗

PubMed1996

Studies of the c-Mpl thrombopoietin receptor through gene disruption and activation.

The c-mpl gene encodes a receptor for thrombopoietin (TPO), a cytokine that potently stimulates megakaryocytopoiesis. To study the mechanisms of c-Mpl activation, we generated constitutively active receptor mutants. Substitution of cysteine residues into a dimer interface homology domain of c-Mpl forced ligand-independent homodimerization and constitutive receptor activation. In factor-dependent cells, mutant receptors induced autonomous growth and tumorigenicity. The receptors were constitutively phosphorylated in these cells, as were signal transduction molecules implicated in Mpl function. These data suggest that the normal process of ligand-induced Mpl activation involves receptor homodimerization and that mutated forms of the cellular mpl gene can contribute to tumorigenicity. We have also examined the biological role of c-Mpl in mpl-deficient mice generated via homologous recombination in embryonic stem cells. Homozygous mutant animals were deficient in megakaryocytes and severely thrombocytopenic. Mature cells from all other hemopoietic lineages were unaffected. Bone marrow cells from mpl-/- mice were incapable of binding to TPO or responding to the cytokine in clonogenic assays, and further displayed a marked deficiency in progenitor cells capable of megakaryocyte colony formation in response to other stimuli. Moreover, total progenitor numbers were also deficient and included significant reductions in colony-forming cells of multiple hemopoietic lineages. Unexpectedly, the numbers of progenitor cells of all lineages were not perturbed in mid-gestation mpl-/- fetal liver. Our analyses suggest an indispensable role for c-Mpl in megakaryocyte development and reveal that the function of TPO and its receptor is not confined solely to activities in megakaryocytopoiesis.

Animals↗

PubMed1999

Structural alterations in the translational attenuator of constitutively expressed ermC genes.

Sequence deletions of 16, 59, and 111 bp as well as a tandem duplication of 272 bp with respect to the corresponding sequence of pT48 were identified in the regulatory regions of constitutively expressed ermC genes. Constitutive ermC gene expression as a consequence of these structural alterations is based on either the prevention of the formation of mRNA secondary structures in the translational attenuator or the preferential formation of those mRNA secondary structures which do not interfere with the translation of the ermC transcripts. A model for the development of sequence deletions in the ermC translational attenuator by homologous recombination is presented and experimentally tested by in vitro selection of constitutively expressed mutants in staphylococcal strains deficient and proficient in homologous recombination.

Animals↗

PubMed2004

Mice deficient for the ets transcription factor elk-1 show normal immune responses and mildly impaired neuronal gene activation.

The transcription factor Elk-1 belongs to the ternary complex factor (TCF) subfamily of Ets proteins. TCFs interact with serum response factor to bind jointly to serum response elements in the promoters of immediate-early genes (IEGs). TCFs mediate the rapid transcriptional response of IEGs to various extracellular stimuli which activate mitogen-activated protein kinase signaling. To investigate physiological functions of Elk-1 in vivo, we generated Elk-1-deficient mice by homologous recombination in embryonic stem cells. These animals were found to be phenotypically indistinguishable from their wild-type littermates. Histological analysis of various tissues failed to reveal any differences between Elk-1 mutant and wild-type mice. Elk-1 deficiency caused no changes in the proteomic displays of brain or spleen extracts. Also, no immunological defects could be detected in mice lacking Elk-1, even upon infection with coxsackievirus B3. In mouse embryonic fibroblasts, Elk-1 was dispensable for c-fos and Egr-1 transcriptional activation upon stimulation with serum, lysophosphatidic acid, or tetradecanoyl phorbol acetate. However, in brains of Elk-1-deficient mice, cortical and hippocampal CA1 expression of c-fos, but not Egr-1 or c-Jun, was markedly reduced 4 h following kainate-induced seizures. This was not accompanied by altered patterns of neuronal apoptosis. Collectively, our data indicate that Elk-1 is essential neither for mouse development nor for adult life, suggesting compensatory activities by other TCFs.

Animals↗

PubMed1996

Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.

Programmed cell death (apoptosis) is a prominent feature of the development of the immune and nervous systems. The identification of the Caenorhabditis elegans cell death gene, ced-3, as a prototype of the interleukin-1beta converting enzyme (ICE) protease family has led to extensive evidence implicating these enzymes in apoptosis. Among the ten or more members of the ICE protease family, CPP32/yama/apopain exhibits the highest similarity to CED-3 in both sequence homology and substrate specificity. To analyse its function in vivo, we generated CPP32-deficient mice by homologous recombination. These mice, born at a frequency lower than expected by mendelian genetics, were smaller than their littermates and died at 1-3 weeks of age. Although their thymocytes retained normal susceptibility to various apoptotic stimuli, brain development in CPP32-deficient mice was profoundly affected, and discernible by embryonic day 12, resulting in a variety of hyperplasias and disorganized cell deployment. These supernumerary cells were postmitotic and terminally differentiated by the postnatal stage. Pyknotic clusters at sites of major morphogenetic change during normal brain development were not observed in the mutant embryos, indicating decreased apoptosis in the absence of CPP32. Thus CPP32 is shown to play a critical role during morphogenetic cell death in the mammalian brain.

Animals↗

PubMed2006

Pot1 deficiency initiates DNA damage checkpoint activation and aberrant homologous recombination at telomeres.

The terminal t-loop structure adopted by mammalian telomeres is thought to prevent telomeres from being recognized as double-stranded DNA breaks by sequestering the 3' single-stranded G-rich overhang from exposure to the DNA damage machinery. The POT1 (protection of telomeres) protein binds the single-stranded overhang and is required for both chromosomal end protection and telomere length regulation. The mouse genome contains two POT1 orthologs, Pot1a and Pot1b. Here we show that conditional deletion of Pot1a elicits a DNA damage response at telomeres, resulting in p53-dependent replicative senescence. Pot1a-deficient cells exhibit overall telomere length and 3' overhang elongation as well as aberrant homologous recombination (HR) at telomeres, manifested as increased telomere sister chromatid exchanges and formation of telomere circles. Telomeric HR following Pot1a loss requires NBS1. Pot1a deletion also results in chromosomal instability. Our results suggest that POT1a is crucial for the maintenance of both telomere integrity and overall genomic stability.

Animals↗

PubMed2001

Deficiency of human BRCA2 leads to impaired homologous recombination but maintains normal nonhomologous end joining.

Carriers of BRCA2 germline mutations are at high risk to develop early-onset breast cancer. The underlying mechanisms of how BRCA2 inactivation predisposes to malignant transformation have not been established. Here, we provide direct functional evidence that human BRCA2 promotes homologous recombination (HR), which comprises one major pathway of DNA double-strand break repair. We found that up-regulated HR after transfection of wild-type (wt) BRCA2 into a human tumor line with mutant BRCA2 was linked to increased radioresistance. In addition, BRCA2-mediated enhancement of HR depended on the interaction with Rad51. In contrast to the tumor suppressor BRCA1, which is involved in multiple DNA repair pathways, BRCA2 status had no impact on the other principal double-strand break repair pathway, nonhomologous end joining. Thus, there exists a specific regulation of HR by BRCA2, which may function to maintain genomic integrity and suppress tumor development in proliferating cells.

BRCA2 Protein↗

PubMed1998

Genetic recombination in Bacillus subtilis 168: effects of recU and recS mutations on DNA repair and homologous recombination.

Bacillus subtilis recombination-deficient mutants were constructed by inserting a selectable marker (cat gene) into the yppB and ypbC coding regions. The yppB:cat and ypbC:cat null alleles rendered cells sensitive to DNA-damaging agents, impaired plasmid transformation (25- and 100-fold), and moderately affected chromosomal transformation when present in an otherwise Rec+ B. subtilis strain. The yppB gene complemented the defect of the recG40 strain. yppB and ypbC and their respective null alleles were termed "recU" and "recU1" (recU:cat) and "recS" and "recS1" (recS:cat), respectively. The recU and recS mutations were introduced into rec-deficient strains representative of the alpha (recF), beta (addA5 addB72), gamma (recH342), and epsilon (recG40) epistatic groups. The recU mutation did not modify the sensitivity of recH cells to DNA-damaging agents, but it did affect inter- and intramolecular recombination in recH cells. The recS mutation did not modify the sensitivity of addAB cells to DNA-damaging agents, and it marginally affected recF, recH, and recU cells. The recS mutation markedly reduced (about 250-fold) intermolecular recombination in recH cells, and there were reductions of 10- to 20-fold in recF, addAB, and recU cells. Intramolecular recombination was blocked in recS recF, recS addAB, and recS recU cells. RecU and RecS have no functional counterparts in Escherichia coli. Altogether, these data indicate that the recU and recS proteins are required for DNA repair and intramolecular recombination and that the recF (alpha epistatic group), addAB (beta), recH (gamma), recU (epsilon), and recS genes provide overlapping activities that compensate for the effects of single mutation. We tentatively placed recS within a new group, termed "zeta".

Bacillus subtilis↗

PubMed2005

Growth defect and mutator phenotypes of RecQ-deficient Neurospora crassa mutants separately result from homologous recombination and nonhomologous end joining during repair of DNA double-strand breaks.

RecQ helicases function in the maintenance of genome stability in many organisms. The filamentous fungus Neurospora crassa has two RecQ homologs, QDE3 and RECQ2. We found that the qde-3 recQ2 double mutant showed a severe growth defect. The growth defect was alleviated by mutation in mei-3, the homolog of yeast RAD51, which is required for homologous recombination (HR), suggesting that HR is responsible for this phenotype. We also found that the qde-3 recQ2 double mutant showed a mutator phenotype, yielding mostly deletions. This phenotype was completely suppressed by mutation of mus-52, a homolog of the human KU80 gene that is required for nonhomologous end joining (NHEJ), but was unaffected by mutation of mei-3. The high spontaneous mutation frequency in the double mutant is thus likely to be due to NHEJ acting on an elevated frequency of double-strand breaks (DSBs) and we therefore suggest that QDE3 and RECQ2 maintain chromosome stability by suppressing the formation of spontaneous DSBs.

Adenosine Triphosphatases↗

PubMed2000

Atm deficiency causes an increased frequency of intrachromosomal homologous recombination in mice.

Ataxia telangiectasia (AT) patients have inactivating mutations in both copies of the ATM gene. The ATM protein that the gene encodes is involved in DNA double-strand break (DSB) recognition; in its absence, p53 response to DSBs is delayed and reduced. In addition, AT patients have a high propensity for cancer, and cells from these patients show chromosomal instability. Here, using an in vivo mouse model system with the pink-eyed unstable mutation, we demonstrate that the absence of functional Atm results in a significantly elevated frequency of intrachromosomal recombination resulting in deletion events (wild-type 17.73%, heterozygous Atm 15.72%, and mutant Atm 30.33%). No such increase was observed in mice heterozygous for Atm. These results further advocate the role of ATM in maintaining genomic integrity after the onset of endogenous damage. This system relies on the initiation of events during a relatively short time frame to produce an observable deletion product. AT patients have a lifelong exposure to endogenous damage and perhaps similarly acting external agents. Because 25% of our genome consists of repeated elements, genomic instability due to an increased level of homologous recombination between such repeats, as observed here, may contribute to carcinogenesis in AT patients.

Animals↗

PubMed2004

Zinc finger gene fez-like functions in the formation of subplate neurons and thalamocortical axons.

fez-like (fezl) is a forebrain-expressed zinc finger gene required for the formation of the hypothalamic dopaminergic and serotonergic (monoaminergic) neurons in zebrafish. To reveal its function in mammals, we analyzed the expression of the mouse orthologue of fezl and generated fezl-deficient mice by homologous recombination. Mouse fezl was expressed specifically in the forebrain from embryonic day 8.5. At mid-gestation, fezl expression was detected in subdomains of the forebrain, including the dorsal telencephalon and ventral diencephalon. Unlike the zebrafish fezl mutant too few, the fezl-deficient mice displayed normal development of hypothalamic monoaminergic neurons, but showed abnormal "hyperactive" behavior. In fezl(-/-) mice, the thalamocortical axons (TCA) were reduced in number and aberrantly projected to the cortex. These mutants had a reduced number of subplate neurons, which are involved in guiding the TCA from the dorsal thalamus, although the subplate neurons were born normally. These results suggest that fezl is required for differentiation or survival of the subplate neurons, and reduction of the subplate neurons in fezl-deficient mice leads to abnormal development of the TCA, providing a possible link between the transcriptional regulation of forebrain development and hyperactive behavior.

Animals↗