RESUMO
Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.
Assuntos
Schizosaccharomyces , Humanos , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMO
The healing of broken chromosomes by de novo telomere addition, while a normal developmental process in some organisms, has the potential to cause extensive loss of heterozygosity, genetic disease, or cell death. However, it is unclear how de novo telomere addition (dnTA) is regulated at DNA double-strand breaks (DSBs). Here, using a non-essential minichromosome in fission yeast, we identify roles for the HR factors Rqh1 helicase, in concert with Rad55, in suppressing dnTA at or near a DSB. We find the frequency of dnTA in rqh1Δ rad55Δ cells is reduced following loss of Exo1, Swi5 or Rad51. Strikingly, in the absence of the distal homologous chromosome arm dnTA is further increased, with nearly half of the breaks being healed in rqh1Δ rad55Δ or rqh1Δ exo1Δ cells. These findings provide new insights into the genetic context of highly efficient dnTA within HR intermediates, and how such events are normally suppressed to maintain genome stability.
Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Reparo de DNA por Recombinação/genética , Proteínas de Schizosaccharomyces pombe/genética , Telômero/genética , Cromossomos Fúngicos/genética , Quebras de DNA de Cadeia Dupla , Exodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico/genética , Instabilidade Genômica/genética , Perda de Heterozigosidade/genética , Rad51 Recombinase/genética , Schizosaccharomyces/genéticaRESUMO
DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.
Assuntos
Quebras de DNA de Cadeia Dupla , Clivagem do DNA , Instabilidade Genômica , Reparo de DNA por Recombinação , Schizosaccharomyces/genética , Pontos de Checagem do Ciclo Celular , Quinase do Ponto de Checagem 2/metabolismo , Cromossomos Fúngicos/genética , Hibridização Genômica Comparativa , Exodesoxirribonucleases/metabolismo , Genoma Fúngico , Perda de Heterozigosidade , Nucleotídeos/biossíntese , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The non-structural protein NS1 of the influenza A virus is a good target for the development of diagnostic assays. In this study, three NS1 monoclonal antibodies (mAbs) were generated by using recombinant NS1 protein of H5N1 virus and found to bind both the native and denatured forms of NS1. Two of the mAbs, 6A4 and 2H6, bind NS1 of three different strains of influenza A virus, namely H1N1, H3N2, and H5N1. Epitope mapping revealed that residues 42-53 of H5N1 NS1 are essential for the interaction with both mAbs. Between the three strains, there is only one amino acid difference in this domain, which is consistent with the observed cross-reactivities. On the other hand, mAb 1G1 binds to residues 206-215 of H5N1 NS1 and does not bind NS1 of H1N1 or H3N2. Furthermore, all three mAbs detected NS1 proteins expressed in virus infected MDCK cells and indirect immunofluorescence staining with mAbs 6A4 and 2H6 provided an alternative method for viral titer determination. Quantifying the numbers of fluorescent foci units yielded viral titers for three different isolates of H5N1 virus that are highly comparable to that obtained by observing cytopathic effect induced by virus infection. Importantly, this alternative method yields results at 1 day post-infection while the conventional method using cytopathic effect yields results at 3 days post-infection. The results showed that this new panel of NS1 antibodies can detect NS1 protein expressed during viral infection and can be used for fast and easy titration of influenza A virus. J. Med. Virol. 82:467-475, 2010. (c) 2010 Wiley-Liss, Inc.
Assuntos
Anticorpos Antivirais , Técnicas de Laboratório Clínico/métodos , Vírus da Influenza A/classificação , Vírus da Influenza A/isolamento & purificação , Influenza Humana/diagnóstico , Proteínas não Estruturais Virais/imunologia , Animais , Anticorpos Monoclonais , Linhagem Celular , Efeito Citopatogênico Viral , Cães , Mapeamento de Epitopos , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A/imunologia , Fatores de Tempo , Virologia/métodosRESUMO
The hemagglutinin (HA) of influenza A virus plays an essential role in mediating the entry of the virus into host cells. Here, recombinant full-length HA5 protein from a H5N1 isolate (A/chicken/hatay/2004(H5N1)) was expressed and purified from the baculovirus-insect cell system. As expected, full-length HA5 elicits strong neutralizing antibodies, as evaluated in micro-neutralization tests using HA5 pseudotyped lentiviral particles. In addition, two fragments of HA5 were expressed in bacteria and the N-terminal fragment, covering the ectodomain before the HA1/HA2 polybasic cleavage site, was found to elicit neutralizing antibodies. But the C-terminal fragment, which covers the remaining portion of the ectodomain, did not. Neutralizing titer of the anti-serum against the N-terminal fragment is only four times lower than the anti-serum against the full-length HA5 protein. Using a novel membrane fusion assay, the abilities of these antibodies to block membrane fusion were found to correlate well with the neutralization activities.
Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Proteínas Recombinantes/imunologia , Animais , Bactérias , Baculoviridae/genética , Linhagem Celular , Vetores Genéticos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Testes de Neutralização , Proteínas Recombinantes/genética , SpodopteraRESUMO
Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.
Assuntos
Quebras de DNA de Cadeia Dupla , Perda de Heterozigosidade/genética , Recombinação Genética , Schizosaccharomyces/genética , Telômero/metabolismo , Translocação Genética , Alelos , Sequência de Bases , Cromossomos Fúngicos/metabolismo , Troca Genética , Reparo do DNA , Marcadores Genéticos , Modelos Genéticos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Mutação/genética , Filogenia , Rad51 Recombinase/metabolismo , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile, a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.