Organization of Early and Late Replicating DNA in Human Chromosome Territories

Self Cite

DNA replication occurs in mammalian cells at so-called replication foci occupying defined nuclear sites at specific times during S phase. It is an unresolved problem how this specific spatiotemporal organization of replication foci is determined. Another unresolved question remains as to what extent DNA is redistributed during S phase. To investigate these problems, we visualized the replicating DNA and the replication machinery simultaneously in living HeLa cells. Time-lapse analyses revealed that DNA was not redistributed to other nuclear sites during S phase. Furthermore, the results showed that DNA is organized into stable aggregates equivalent to replication foci. These aggregates, which we call sub-chromosomal foci, stably maintained their replication timing from S phase to S phase. During S-phase progression, the replication machinery sequentially proceeded through spatially adjacent sets of sub-chromosomal foci. These findings imply that the specific nuclear substructure of chromosomes and the order of their stable subunits determine the spatiotemporal organization of DNA replication.

Section: Discussionmentioning

confidence: 99%

Stable chromosomal units determine the spatial and temporal organization of DNA replication

Sadoni

Cardoso

Stelzer

et al. 2004

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“…One replicon cluster might approximately correspond to 1 Mb (Berezney et al, 2000;Berezney, 2002). Furthermore, previous works have characterized 1Mb domains as units of higher-order chromosome organization and coordinated replication (Zink et al, 1998;Zink et al, 1999).…”

Section: Discussionmentioning

confidence: 99%

Macroscopic folding and replication of the homogeneously staining region in late S phase leads to the appearance of replication bands in mitotic chromosomes

Shimizu

Shingaki

2004

The chromosomal G/R bands are alternating domains differing in their nucleotide sequence biases. The bands are also related to the time of replication: pulse-labeling during S phase makes the replication sites as visible as replication bands that are close to the G/R bands in mitotic chromosomes. We previously showed that a plasmid bearing a mammalian replication origin efficiently generated a chromosomal homogeneously staining region (HSR). Here, we analyze the replication of this artificial HSR and show that it was replicated at the last stage of S phase. The HSR was composed of plasmid repeats only; nonetheless, we found that replication sites pulse-labeled during late S phase appeared as bands in the mitotic HSR and their number was dependent on the length of the HSR. Therefore, replication bands might not arise from sequence information per se. To understand the chronological order of appearance of replication sites, we performed a double pulse-chase experiment using IdU and CldU. Replication of the entire HSR required 100-120 minutes. During this period, the replicated sites appeared as bands at the first and last stages, but in between were apparently scattered along the entire HSR. An analysis of S-phase nuclei revealed that the replication started at the periphery of the globular HSR domain, followed by initiation in the internal domain. The replicated HSR appeared as a ring or a pair of extended spirals in late G2-phase nuclei. To account for these findings, we present a model in which the HSR is folded as a coiled-coil structure that is replicated from the outside to the inside in S phase nuclei.

“…Functional importance has been assigned to this territorial organization of chromatin, on the assumption that genomic sequences inside domains are less accessible to regulatory factors compared with those at the surface Cremer et al, 1993;Kurz et al, 1996;Scheuermann et al, 2004;Cremer et al, 2006). Apart from this 'processive' compaction of chromatin, replication timing reveals another type of pattern: Isochronously replicating domains stably persist through cell divisions (Jackson and Pombo, 1998;Zink et al, 1999;Zink, 2006). A third type of chromatin organization is reflected by the concentration of more densely packed chromatin in perinuclear and peri-nucleolar regions.…”

Section: Introductionmentioning

confidence: 99%

Experimental evidence for the influence of molecular crowding on nuclear architecture

Richter

Neßling

Lichter

2007

112

124

Many compounds in the cell nucleus are structurally organized. To assess the influence of structural organization on nuclear function, we investigated the physical mechanisms of structure formation by using molecular crowding as a parameter for nuclear integrity. Molecular crowding promotes compaction of macromolecular compounds depending on their size and shape without the need for site-specific interactions. HeLa and MCF7 cells were incubated with hypertonic medium to increase crowding of their macromolecular content as a result of the osmotic loss of water. Supplementation of sucrose, sorbitol or NaCl to the growth medium shifted nuclear organization, observed by fluorescence and electron microscopy, towards compaction of chromatin and segregation of other nuclear compounds. With increasing hypertonic load and incubation time, this nuclear re-organization proceeded gradually, irrespective of the substances used, and reversibly relaxed to a regular phenotype upon re-incubation of cells in isotonic growth medium. Gradual and reversible re-organization are major features of controlled de-mixing by molecular crowding. Of fundamental importance for nuclear function, we discuss how macromolecular crowding could account for the stabilization of processes that involve large, macromolecular machines.