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Biomedical subjects

André Nussenzweig

Publications and source records attributed to André Nussenzweig.

At least 19 recordsLinked to original sources
PubMed2007

Enhanced intra-switch region recombination during immunoglobulin class switch recombination in 53BP1-/- B cells.

Immunoglobulin class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. U:G mismatches created by AID are processed to produce lesions that recruit and activate DNA damage response proteins including Ataxia-telangiectasia mutated (ATM), histone H2AX, Nijmegen breakage syndrome 1 (Nbs1), and p53 binding protein 1 (53BP1). Among these proteins, absence of 53BP1 produces the most severe impairment of class switching. Here, we demonstrate that AID is targeted normally to switch region DNA and that intra-switch region recombination is enhanced in 53BP1-/- B cells. In addition, Smicro-Sgamma1 switch region junctions cloned from 53BP1-/- B cells show unusual insertions suggestive of failed class switching. Our data are consistent with a role for 53BP1 in stabilizing the synapsis of switch regions during CSR.

Animals↗
PubMed2006

Spatio-temporal dynamics of chromatin containing DNA breaks.

The cellular response to DNA breaks consists of a complex signaling network that coordinates the initial recognition of the lesion with the induction of cell cycle checkpoints and DNA repair. With DNA wrapped around histone proteins and packaged into higher order levels of chromatin structure, the detection of a single DNA break (DSB) in the genome is the molecular equivalent of finding a needle in a haystack. A recent study from our laboratory used high-resolution electron microscropy and live cell imaging to demonstrate that chromatin undergoes a marked reorganization in response to a DSB. In an energy dependent manner, chromatin rapidly decondenses to a more open configuration in the regions surrounding the lesion. We propose that this ATP dependent chromatin-remodeling event facilitates the subsequent recognition and processing of damaged DNA. While the chromatin surrounding the lesion remodels to a more open configuration, the DNA break itself remains relatively immobile over time, consistent with the idea that DNA damage response proteins migrate to positionally stable sites of damaged DNA. The lack of significant movement of chromatin regions containing DSBs has implications for the process by which chromosomal translocations form.

Animals↗
PubMed2006

Switching on chromosomal translocations.

Activation-induced deaminase initiates three different antibody diversification reactions: class switch recombination, somatic hypermutation (SHM), and gene conversion. We have shown that, in addition to antibody diversification, activation-induced deaminase can also initiate Burkitt's lymphoma-like c-myc/IgH translocations. However, distinct DNA damage- and oncogene-induced checkpoints operate in B cells to produce a normal intrachromosomal class switch recombination event or an aberrant interchromosomal fusion. These findings open the way to study the molecular pathways taking place at the early stages of malignant transformation.

Cell Transformation, Neoplastic↗
PubMed2006

Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo.

The ATM (ataxia telangiectasia mutated) protein kinase is activated under physiological and pathological conditions that induce DNA double-strand breaks (DSBs). Loss of ATM or failure of its activation in humans and mice lead to defective cellular responses to DSBs, such as cell cycle checkpoints, radiation sensitivity, immune dysfunction, infertility and cancer predisposition. A widely used biological marker to identify the active form of ATM is the autophosphorylation of ATM at a single, conserved serine residue (Ser 1981 in humans; Ser 1987 in mouse). Here we show that Atm-dependent responses are functional at the organismal and cellular level in mice that express a mutant form of Atm (mutation of Ser to Ala at position 1987) as their sole Atm species. Moreover, the mutant protein does not exhibit dominant-negative interfering activity when expressed physiologically or overexpressed in the context of Atm heterozygous mice. These results suggest an alternative mode for stimulation of Atm by DSBs in which Atm autophosphorylation at Ser 1987, like trans-phosphorylation of downstream substrates, is a consequence rather than a cause of Atm activation.

Animals↗
PubMed2006

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

Adenosine Triphosphate↗
PubMed2006

MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals.

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Ku86 preserves chromatin integrity in cells adapted to high NaCl.

Cells adapted to high NaCl have many DNA breaks both in cell culture and in the renal inner medulla in vivo; yet they survive, function, and even proliferate. Here, we show that Ku86 is important for maintaining chromosomal integrity despite the continued presence of DNA breaks. The Ku heterodimer is part of DNA-dependent PK (DNA-PK), a complex that contributes by nonhomologous end joining to repair of double-strand breaks. We demonstrate that cells deficient in Ku86, but not cells deficient in DNA-PKcs (the catalytic subunit of DNA-PK), are hypersensitive to high NaCl as manifested by profound inhibition of proliferation, aberrant mitosis, and increased chromosomal fragmentation. Lower eukaryotes, including the soil nematode Caenorhabditis elegans, lack a DNA-PKcs homologue but are able to adapt to high NaCl. We show that cells of C. elegans adapted to high NaCl have many DNA breaks, similar to the mammalian cells adapted to high NaCl. Ku86 mutant C. elegans as well as C. elegans fed with cku86 dsRNA also display hypersensitivity to high NaCl, characterized by a reduced number of progeny and prolonged generation time in high NaCl. We propose that Ku86 ameliorates the effects of high NaCl-induced DNA breaks in adapted cells by supporting alignment of the broken ends of the DNA and thus maintaining integrity of the fragmented chromatin.

Adaptation, Physiological↗
PubMed2005

Role of Nbs1 in the activation of the Atm kinase revealed in humanized mouse models.

Nijmegen breakage syndrome (NBS) is a chromosomal fragility disorder that shares clinical and cellular features with ataxia telangiectasia. Here we demonstrate that Nbs1-null B cells are defective in the activation of ataxia-telangiectasia-mutated (Atm) in response to ionizing radiation, whereas ataxia-telangiectasia- and Rad3-related (Atr)-dependent signalling and Atm activation in response to ultraviolet light, inhibitors of DNA replication, or hypotonic stress are intact. Expression of the main human NBS allele rescues the lethality of Nbs1-/- mice, but leads to immunodeficiency, cancer predisposition, a defect in meiotic progression in females and cell-cycle checkpoint defects that are associated with a partial reduction in Atm activity. The Mre11 interaction domain of Nbs1 is essential for viability, whereas the Forkhead-associated (FHA) domain is required for T-cell and oocyte development and efficient DNA damage signalling. Reconstitution of Nbs1 knockout mice with various mutant isoforms demonstrates the biological impact of impaired Nbs1 function at the cellular and organismal level.

ATP-Binding Cassette Transporters↗
PubMed2005

Genomic instability, endoreduplication, and diminished Ig class-switch recombination in B cells lacking Nbs1.

Mre11, Rad50, and Nbs1 form an evolutionarily conserved protein complex (Mre11-Rad50-Nbs1, MRN) that has been proposed to function as a DNA damage sensor. Hypomorphic mutations in Mre11 and Nbs1 result in the human ataxia-telangiectasia-like disorder and Nijmegen breakage syndrome (NBS), respectively. In contrast, complete inactivation of Mre11, Rad50, or Nbs1 leads to early embryonic lethality, suggesting that the hypomorphic mutations may fail to reveal some of the essential functions of MRN. Here, we use Cre-loxP-mediated recombination to restrict Nbs1 deletion to B lymphocytes. We find that disruption of Nbs1 results in the accumulation of high levels of spontaneous DNA damage, impaired proliferation, and chromosomal endoreduplication. Moreover, we show that Ig class-switch recombination (CSR) is diminished in Nbs1-deficient B cells. The CSR defect is B cell-intrinsic, independent of switch-region transcription, and a consequence of inefficient recombination at the DNA level. Our findings reveal that Nbs1 is critical for efficient Ig CSR and maintenance of the integrity of chromosomal structure and number.

Animals↗
PubMed2005

Specific association of mouse MDC1/NFBD1 with NBS1 at sites of DNA-damage.

Human MDC1/NFBD1 has been found to interact with key players of the DNA-damage response machinery. Here, we identify and describe a functional homologue of MDC1/ NFBD1 in Mus musculus. The mouse homologue, mMDC1, retains the key motifs identified in the human protein and in response to ionizing radiation forms foci that co-localize with the MRE11-RAD50-NBS1 (MRN) complex and factors such as gammaH2AX and 53BP1. In addition, mMDC1 is associated with DNA damage sites generated during meiotic recombination as well as the X and Y chromosomes during the late stages of meiotic prophase I. Finally, whereas MDC1 shows strong colocalization with the MRN complex in response to DNA damage it does not co-localize with the MRN complex on replicating chromatin. These data suggest that mMDC1 is a marker for both exogenously and endogenously generated DNA double-stranded breaks and that its interaction with the MRN complex is initiated exclusively by DNA damage.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Silencing of unsynapsed meiotic chromosomes in the mouse.

In Neurospora, DNA unpaired in meiosis both is silenced and induces silencing of all DNA homologous to it. This process, called meiotic silencing by unpaired DNA, is thought to protect the host genome from invasion by transposable elements. We now show that silencing of unpaired (unsynapsed) chromosome regions also takes place in the mouse during both male and female meiosis. The tumor suppressor protein BRCA1 is implicated in this silencing, mirroring its role in the meiotic silencing of the X and Y chromosomes in normal male meiosis. These findings impact on the interpretation of the relationship between synaptic errors and sterility in mammals and extend our understanding of the biology of Brca1.

Animals↗
PubMed2004

ATM is required for efficient recombination between immunoglobulin switch regions.

Ataxia telangiectasia mutated (ATM) kinase is critical for initiating the signaling pathways that lead to cell cycle checkpoints and DNA double strand break repair. In the absence of ATM, humans and mice show a primary immunodeficiency that includes low serum antibody titers, but the role of ATM in antigen-driven immunoglobulin gene diversification has not been defined. Here, we show that although ATM is dispensable for somatic hypermutation, it is required for efficient class switch recombination (CSR). The defect in CSR is not due to alterations in switch region transcription, accessibility, DNA damage checkpoint protein recruitment, or short-range intra-switch region recombination. Only long-range inter-switch recombination is defective, indicating an unexpected role for ATM in switch region synapsis during CSR.

Animals↗
PubMed2004

Functional interaction between BLM helicase and 53BP1 in a Chk1-mediated pathway during S-phase arrest.

Bloom's syndrome is a rare autosomal recessive genetic disorder characterized by chromosomal aberrations, genetic instability, and cancer predisposition, all of which may be the result of abnormal signal transduction during DNA damage recognition. Here, we show that BLM is an intermediate responder to stalled DNA replication forks. BLM colocalized and physically interacted with the DNA damage response proteins 53BP1 and H2AX. Although BLM facilitated physical interaction between p53 and 53BP1, 53BP1 was required for efficient accumulation of both BLM and p53 at the sites of stalled replication. The accumulation of BLM/53BP1 foci and the physical interaction between them was independent of gamma-H2AX. The active Chk1 kinase was essential for both the accurate focal colocalization of 53BP1 with BLM and the consequent stabilization of BLM. Once the ATR/Chk1- and 53BP1-mediated signal from replicational stress is received, BLM functions in multiple downstream repair processes, thereby fulfilling its role as a caretaker tumor suppressor.

Bloom Syndrome↗
PubMed2004

AID is required for c-myc/IgH chromosome translocations in vivo.

Chromosome translocations between c-myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-myc/IgH chromosome translocations induced by IL6.

Animals↗
PubMed2004

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair.

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.

HeLa Cells↗
PubMed2004

Phosphorylation of histone H2B at DNA double-strand breaks.

Posttranslational modifications of histone tails regulate numerous biological processes including transcription, DNA repair, and apoptosis. Although recent studies suggest that structural alterations in chromatin are critical for triggering the DNA damage response, very little is known about the nature of DNA damage-induced chromatin perturbations. Here we show that the serine 14 residue in the NH(2)-terminal tail of histone H2B is rapidly phosphorylated at sites of DNA double-strand breaks. At late time points after irradiation, the phosphorylated form of H2B, H2B-(Ser14P), accumulates into irradiation-induced foci. H2B-(Ser14P) foci formation is not associated with the apoptotic phosphorylation of H2B but is strictly dependent on the phosphorylated isoform of H2AX. Our results broaden the spectrum of histone modifications that constitute the DNA damage "histone code" and suggest a model for the underlying chromatin structure within damage-induced foci.

Amino Acid Substitution↗
PubMed2004

Collaboration of homologous recombination and nonhomologous end-joining factors for the survival and integrity of mice and cells.

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are mechanistically distinct DNA repair pathways that contribute substantially to double-strand break (DSB) repair in mammalian cells. We have combined mutations in factors from both repair pathways, the HR protein Rad54 and the DNA-end-binding factor Ku80, which has a role in NHEJ. Rad54(-/-)Ku80(-/-) mice were severely compromised in their survival, such that fewer double mutants were born than expected, and only a small proportion of those born reached adulthood. However, double-mutant mice died at lower frequency from tumors than Ku80 single mutant mice, likely as a result of rapid demise at a young age from other causes. When challenged with an exogenous DNA damaging agent, ionizing radiation, double-mutant mice were exquisitely sensitive to low doses. Tissues and cells from double-mutant mice also showed indications of spontaneous DNA damage. Testes from some Rad54(-/-)Ku80(-/-) mice displayed enhanced apoptosis and reduced sperm production, and embryonic fibroblasts from Rad54(-/-)Ku80(-/-) animals accumulated foci of gamma-H2AX, a marker for DSBs. The substantially increased DNA damage response in the double mutants implies a cooperation of the two DSB repair pathways for survival and genomic integrity in the animal.

Aging↗