RESUMO
Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. Here we show that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes.
Assuntos
Citoesqueleto de Actina/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Heterocromatina/metabolismo , Movimento , Miosinas/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Heterocromatina/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Chaperonas Moleculares , Reparo de DNA por RecombinaçãoRESUMO
Heterochromatin is mostly composed of long stretches of repeated DNA sequences prone to ectopic recombination during double-strand break (DSB) repair. In Drosophila, "safe" homologous recombination (HR) repair of heterochromatic DSBs relies on a striking relocalization of repair sites to the nuclear periphery. Central to understanding heterochromatin repair is the ability to investigate the 4D dynamics (movement in space and time) of repair sites. A specific challenge of these studies is preventing phototoxicity and photobleaching effects while imaging the sample over long periods of time, and with sufficient time points and Z-stacks to track repair foci over time. Here we describe an optimized approach for high-resolution live imaging of heterochromatic DSBs in Drosophila cells, with a specific emphasis on the fluorescent markers and imaging setup used to capture the motion of repair foci over long-time periods. We detail approaches that minimize photobleaching and phototoxicity with a DeltaVision widefield deconvolution microscope, and image processing techniques for signal recovery postimaging using SoftWorX and Imaris software. We present a method to derive mean square displacement curves revealing some of the biophysical properties of the motion. Finally, we describe a method in R to identify tracts of directed motions (DMs) in mixed trajectories. These approaches enable a deeper understanding of the mechanisms of heterochromatin dynamics and genome stability in the three-dimensional context of the nucleus and have broad applicability in the field of nuclear dynamics.
Assuntos
Drosophila/genética , Heterocromatina/metabolismo , Microscopia de Fluorescência/métodos , Reparo de DNA por Recombinação , Software , Animais , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Drosophila/metabolismo , Heterocromatina/genética , Imageamento Tridimensional/métodosRESUMO
Heterochromatin is mostly composed of repeated DNA sequences prone to aberrant recombination. How cells maintain the stability of these sequences during double-strand break (DSB) repair has been a long-standing mystery. Studies in Drosophila cells revealed that faithful homologous recombination repair of heterochromatic DSBs relies on the striking relocalization of repair sites to the nuclear periphery before Rad51 recruitment and repair progression. Here, we summarize our current understanding of this response, including the molecular mechanisms involved, and conserved pathways in mammalian cells. We will highlight important similarities with pathways identified in budding yeast for repair of other types of repeated sequences, including rDNA and short telomeres. We will also discuss the emerging role of chromatin composition and regulation in heterochromatin repair progression. Together, these discoveries challenged previous assumptions that repair sites are substantially static in multicellular eukaryotes, that heterochromatin is largely inert in the presence of DSBs, and that silencing and compaction in this domain are obstacles to repair.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.
Assuntos
Núcleo Celular/genética , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Saccharomyces cerevisiae/genética , Animais , Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Aging is characterized by genome instability, which contributes to cancer formation and cell lethality leading to organismal decline. The high levels of DNA double-strand breaks (DSBs) observed in old cells and premature aging syndromes are likely a primary source of genome instability, but the underlying cause of their formation is still unclear. DSBs might result from higher levels of damage or repair defects emerging with advancing age, but repair pathways in old organisms are still poorly understood. Here, we show that premeiotic germline cells of young and old flies have distinct differences in their ability to repair DSBs by the error-free pathway homologous recombination (HR). Repair of DSBs induced by either ionizing radiation (IR) or the endonuclease I-SceI is markedly defective in older flies. This correlates with a remarkable reduction in HR repair measured with the DR-white DSB repair reporter assay. Strikingly, most of this repair defect is already present at 8 days of age. Finally, HR defects correlate with increased expression of early HR components and increased recruitment of Rad51 to damage in older organisms. Thus, we propose that the defect in the HR pathway for germ cells in older flies occurs following Rad51 recruitment. These data reveal that DSB repair defects arise early in the aging process and suggest that HR deficiencies are a leading cause of genome instability in germ cells of older animals.
Assuntos
Envelhecimento/fisiologia , Quebras de DNA de Cadeia Dupla , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Células Germinativas/metabolismo , Recombinação Homóloga/genética , Animais , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/efeitos da radiação , Células Germinativas/citologia , Células Germinativas/efeitos da radiação , Meiose/efeitos da radiação , Modelos Biológicos , Rad51 Recombinase/metabolismo , Radiação IonizanteRESUMO
Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, 'safe' homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. Here we show that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote.
Assuntos
Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Heterocromatina/genética , Reparo de DNA por Recombinação , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Heterocromatina/metabolismo , Hibridização in Situ Fluorescente , Mutação , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Imagem com Lapso de Tempo/métodosAssuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Reparo do DNA/fisiologia , Proteínas de Drosophila/metabolismo , Heterocromatina/metabolismo , Chaperonas Moleculares/metabolismo , Cromossomos Politênicos/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Quebras de DNA de Cadeia Dupla , Drosophila melanogaster , Instabilidade Genômica/fisiologia , Miosinas/metabolismoRESUMO
The animal sperm nucleus is characterized by an extremely compacted organization of its DNA after the global replacement of histones with sperm-specific nuclear basic proteins, such as protamines. In the absence of DNA repair activity in the mature gamete, the integrity of the paternal genome is potentially challenged by the unique topological constraints exerted on sperm DNA. In addition, the maintenance of paternal DNA integrity during the rapid remodeling of sperm chromatin at fertilization has long been regarded as a maternal trait. However, little is known about the nature of the egg proteins involved in this essential aspect of zygote formation. We had previously characterized the unique phenotype of the classical Drosophila maternal effect mutant maternal haploid (mh), which specifically affects the integration of paternal chromosomes in the zygote. Here we show that MH is the fly ortholog of the recently identified human DVC1/Spartan protein, a conserved regulator of DNA damage tolerance. Like Spartan, MH protein is involved in the resistance to UV radiation and recruits the p97/TER94 segregase to stalled DNA replication forks in somatic cells. In the zygote, we found that the mh phenotype is consistent with perturbed or incomplete paternal DNA replication. Remarkably, however, the specific accumulation of MH in the male pronucleus before the first S phase suggests that this maternal protein is required to maintain paternal DNA integrity during nuclear decondensation or to set the paternal chromatin landscape in preparation of the first zygotic cycle.
Assuntos
Cromossomos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Animais , Replicação do DNA , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Haploidia , Zigoto/metabolismoRESUMO
BACKGROUND: A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, HipHop. We previously showed that the hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. RESULTS: Here we show that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and is retained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, we demonstrate by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. CONCLUSIONS: Our data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. We propose that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes.