RESUMO
Since the pioneering proposal of the replicon model of DNA replication 50 years ago, the predicted replicons have not been identified and quantified at the cellular level. Here, we combine conventional and super-resolution microscopy of replication sites in live and fixed cells with computational image analysis. We complement these data with genome size measurements, comprehensive analysis of S-phase dynamics and quantification of replication fork speed and replicon size in human and mouse cells. These multidimensional analyses demonstrate that replication foci (RFi) in three-dimensional (3D) preserved somatic mammalian cells can be optically resolved down to single replicons throughout S-phase. This challenges the conventional interpretation of nuclear RFi as replication factories, that is, the complex entities that process multiple clustered replicons. Accordingly, 3D genome organization and duplication can be now followed within the chromatin context at the level of individual replicons.
Assuntos
Cromatina/ultraestrutura , Replicação do DNA , Replicon , Fase S/genética , Animais , Linhagem Celular , Cromatina/química , Cromatina/metabolismo , Expressão Gênica , Tamanho do Genoma , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Cinética , Camundongos , Imagem Molecular , Mioblastos/metabolismo , Mioblastos/ultraestrutura , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Especificidade da EspécieRESUMO
We studied the nuclear topography of RNA transcription and DNA replication in mammalian cell types with super-resolution fluorescence microscopy, which offers a resolution beyond the classical Abbe/Raleigh limit. Three-dimensional structured illumination microscopy (3D-SIM) demonstrated a network of channels and wider lacunas, called the interchromatin compartment (IC). The IC starts at nuclear pores and expands throughout the nuclear space. It is demarcated from the compact interior of higher-order chromatin domains (CDs) by a 100-200-nm thick layer of decondensed chromatin, termed the perichromatin region (PR). Nascent DNA, nascent RNA, RNA polymerase II (RNA Pol II), as well as histone modifications for transcriptionally competent/active chromatin, are highly enriched in the PR, whereas splicing speckles are observed in the interior of the IC. In line with previous electron microscopic evidence, spectral precision distance/position determination microscopy (SPDM) confirmed the presence of RNA Pol II clusters indicative of transcription factories. Still, a substantial part of transcription apparently takes place outside of such factories. Previous electron microscopic evidence has suggested that the functional nuclear organization of DNA replication depends on brownian movements of chromatin between the CD interior and the PR. As an incentive for future studies, we hypothesize that such movements also take place during transcription, i.e., only the actually transcribed part of a gene may be located within the PR, whereas its major part, including previously or later transcribed sequences, is embedded in a higher-order chromatin configuration in the interior of the CD.
Assuntos
Compartimento Celular , Cromatina/química , Cromatina/metabolismo , Replicação do DNA/genética , Transcrição Gênica , Animais , DNA/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Microscopia , Matriz Nuclear/metabolismo , Processamento de Proteína Pós-Traducional , RNA/química , RNA Polimerase II/química , Splicing de RNA/genéticaRESUMO
DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses.
Assuntos
Replicação do DNA , Microscopia/métodos , Animais , Bromodesoxiuridina/análise , Linhagem Celular , Estruturas do Núcleo Celular/ultraestrutura , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia Confocal , Antígeno Nuclear de Célula em Proliferação/análiseRESUMO
Chromosome-specific paint probes provide a powerful tool with wide applications in cytogenetic analysis. Here, we present a new approach using UV-laser microbeam microdissection in combination with laser-pressure catapulting, which allows the fast isolation of single chromosomes for the generation of chromosome-specific paint probes. To demonstrate the feasibility of this approach, single chromosomes were collected and amplified with degenerate oligonucleotide-primed PCR, hapten-labeled and hybridized onto normal metaphase spreads. Fluorescence in situ hybridization signals revealed specific painting of the respective chromosomes.