RESUMO
While the molecular repertoire of the homologous recombination pathways is well studied, the search mechanism that enables recombination between distant homologous regions is poorly understood. Earlier work suggests that the recombinase RecA, an essential component for homology search, forms an elongated filament, nucleating at the break site. How this RecA structure carries out long-distance search remains unclear. Here, we follow the dynamics of RecA after induction of a single double-strand break on the Caulobacter chromosome. We find that the RecA-nucleoprotein filament, once formed, rapidly translocates in a directional manner in the cell, undergoing several pole-to-pole traversals, until homology search is complete. Concomitant with translocation, we observe dynamic variation in the length of the filament. Importantly in vivo, the RecA filament alone is incapable of such long-distance movement; both translocation and associated length variations are contingent on action of structural maintenance of chromosome (SMC)-like protein RecN, via its ATPase cycle. In summary, we have uncovered the three key elements of homology search driven by RecN: mobility of a finite segment of RecA, changes in filament length, and ability to conduct multiple pole-to-pole traversals, which together point to an optimal search strategy.
Assuntos
Proteínas de Bactérias , Recombinases Rec A , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Cromossomos/metabolismo , DNA de Cadeia SimplesRESUMO
BACKGROUND: The 3D organization of the chromatin fiber in cell nuclei plays a key role in the regulation of gene expression. Genome-wide techniques to score DNA-DNA contacts, such as Hi-C, reveal the partitioning of chromosomes into epigenetically defined active and repressed compartments and smaller "topologically associated" domains. These domains are often associated with chromatin loops, which largely disappear upon removal of cohesin. Because most Hi-C implementations average contact frequencies over millions of cells and do not provide direct spatial information, it remains unclear whether and how frequently chromatin domains and loops exist in single cells. RESULTS: We combine 3D single-molecule localization microscopy with a low-cost fluorescence labeling strategy that does not denature the DNA, to visualize large portions of single human chromosomes in situ at high resolution. In parallel, we develop multi-scale, whole nucleus polymer simulations, that predict chromatin structures at scales ranging from 5 kb up to entire chromosomes. We image chromosomes in G1 and M phase and examine the effect of cohesin on interphase chromatin structure. Depletion of cohesin leads to increased prevalence of loose chromatin stretches, increased gyration radii, and reduced smoothness of imaged chromatin regions. By comparison to model predictions, we estimate that 6-25 or more purely cohesin-dependent chromatin loops coexist per megabase of DNA in single cells, suggesting that the vast majority of the genome is enclosed in loops. CONCLUSION: Our results provide new constraints on chromatin structure and showcase an affordable non-invasive approach to study genome organization in single cells.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos/genética , Modelos Biológicos , Núcleo Celular/metabolismo , Cromatina/metabolismo , Células HCT116 , Humanos , Interfase , Mitose , CoesinasRESUMO
In order to replicate, human immunodeficiency virus (HIV-1) reverse-transcribes its RNA genome into DNA, which subsequently integrates into host cell chromosomes. These two key events of the viral life cycle are commonly viewed as separate not only in time, but also in cellular space, since reverse transcription (RT) is thought to be completed in the cytoplasm before nuclear import and integration. However, the spatiotemporal organization of the early viral replication cycle in macrophages, the natural non-dividing target cells that constitute reservoirs of HIV-1 and an obstacle to curing AIDS, remains unclear. Here, we demonstrate that infected macrophages display large nuclear foci of viral DNA (vDNA) and viral RNA, in which multiple viral genomes cluster together. These clusters form in the absence of chromosomal integration, sequester the paraspeckle protein CPSF6, and localize to nuclear speckles. Surprisingly, these viral RNA clusters consist mostly of genomic, incoming RNA, both in cells where reverse transcription is pharmacologically suppressed and in untreated cells. We demonstrate that following temporary inhibition, reverse transcription can resume in the nucleus and lead to vDNA accumulation in these clusters. We further show that nuclear reverse transcription can result in transcription-competent viral DNA. These findings change our understanding of the early HIV-1 replication cycle and may have implications for addressing HIV-1 persistence.
Assuntos
Núcleo Celular/virologia , Genoma Viral/genética , HIV-1/genética , Macrófagos/virologia , Transcrição Reversa/genética , Transporte Ativo do Núcleo Celular/genética , Linhagem Celular , Análise por Conglomerados , Citoplasma/virologia , DNA Viral/genética , Células HEK293 , Infecções por HIV/virologia , Humanos , RNA Viral/genética , Células THP-1 , Replicação Viral/genéticaRESUMO
In our cells, DNA is folded and packed with the help of many proteins into chromatin whose basic unit is a nucleosome-DNA wrapped around octamer of histone proteins. The chain of nucleosomes is further folded and arranged into many layers and has a dynamic organization. How does the complex chromatin organization emerge from interactions among DNA, histones, and non-histone proteins have been a question of great interest. Here we review recent literature that investigated how nucleosome positioning and nucleosome-mediated interactions drive chromatin organization. Unlike our earlier understanding, chromatin is organized into 3D domains of various sizes having irregularly organized nucleosomes. These domains emerge due to heterogeneous nucleosome positioning and diverse inter-nucleosome interactions that vary in space and time.
Assuntos
Montagem e Desmontagem da Cromatina , Nucleossomos , Cromatina , DNA/genética , Histonas/metabolismoRESUMO
The genetic information that instructs transcription and other cellular functions is carried by the chromosomes, polymers of DNA in complex with histones and other proteins. These polymers are folded inside nuclei five orders of magnitude smaller than their linear length, and many facets of this folding correlate with or are causally related to transcription and other cellular functions. Recent advances in sequencing and imaging-based techniques have enabled new views into several layers of chromatin organization. These experimental findings are accompanied by computational modeling efforts based on polymer physics that can provide mechanistic insights and quantitative predictions. Here, we review current knowledge of the main levels of chromatin organization, from the scale of nucleosomes to the entire nucleus, our current understanding of their underlying biophysical and molecular mechanisms, and some of their functional implications.
Assuntos
Cromatina , Genoma , Animais , Biofísica , Núcleo Celular/genética , Simulação por Computador , HumanosRESUMO
DNA double-strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome-wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Histonas/metabolismo , Saccharomycetales/genética , Bleomicina/farmacologia , DNA Fúngico/genética , Mutagênicos/farmacologia , Fosforilação , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/metabolismoRESUMO
It is being increasingly realized that nucleosome organization on DNA crucially regulates DNA-protein interactions and the resulting gene expression. While the spatial character of the nucleosome positioning on DNA has been experimentally and theoretically studied extensively, the temporal character is poorly understood. Accounting for ATPase activity and DNA-sequence effects on nucleosome kinetics, we develop a theoretical method to estimate the time of continuous exposure of binding sites of non-histone proteins (e.g. transcription factors and TATA binding proteins) along any genome. Applying the method to Saccharomyces cerevisiae, we show that the exposure timescales are determined by cooperative dynamics of multiple nucleosomes, and their behavior is often different from expectations based on static nucleosome occupancy. Examining exposure times in the promoters of GAL1 and PHO5, we show that our theoretical predictions are consistent with known experiments. We apply our method genome-wide and discover huge gene-to-gene variability of mean exposure times of TATA boxes and patches adjacent to TSS (+1 nucleosome region); the resulting timescale distributions have non-exponential tails.
Assuntos
Proteínas de Ligação a DNA/genética , Nucleossomos/genética , Ligação Proteica/genética , Transcrição Gênica , Fosfatase Ácida/genética , Fosfatase Ácida/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Regulação da Expressão Gênica , Cinética , Nucleossomos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
We investigate how DNA sequence, ATP-dependent chromatin remodeling and nucleosome-depleted 'barriers' co-operate to determine the kinetics of nucleosome organization, in a stochastic model of nucleosome positioning and dynamics. We find that 'statistical' positioning of nucleosomes against 'barriers', hypothesized to control chromatin structure near transcription start sites, requires active remodeling and therefore cannot be described using equilibrium statistical mechanics. We show that, unlike steady-state occupancy, DNA site exposure kinetics near a barrier is dominated by DNA sequence rather than by proximity to the barrier itself. The timescale for formation of positioning patterns near barriers is proportional to the timescale for active nucleosome eviction. We also show that there are strong gene-to-gene variations in nucleosome positioning near barriers, which are eliminated by averaging over many genes. Our results suggest that measurement of nucleosome kinetics can reveal information about sequence-dependent regulation that is not apparent in steady-state nucleosome occupancy.