ИСТИНА

Background: DNA replication represents one of the essential and most complicated biological processes in proliferating eukaryotic cell life. It is known that in the nucleus, DNA is wrapped around histones to form nucleosomes and other higher order compact chromatin structures. This particular way of genome packing creates certain topological problems during DNA synthesis in the limited nuclear volume. The main idea of our research consists in studies of replication events at the level of chromatin structure visualized in context of native chromatin structure preservation. Materials and methods: First, we examined chromatin organization in active replication sites at various periods of S-phase at ultrastructural level in cell lines expressing GFP-PCNA by immunoelectron microscopy. Next, we studied post replication remodeling of higher order chromatin domains using DNA metabolic pulse labeling with etynyldeoxyuridine (EDU), fixed cells at different time intervals and detected replicated loci with click-chemistry and immunoelectron microscopy. Results: It turns out that in many cases replication sites look like segments of high order chromatin fibrils (chromonema) 200-300 nm thick. Using pre-embedding labeling technique, we found preferential peripheral location of the label in active replication sites. While overall structure of replicating loci did not change compared to neighboring unreplicated chromatin domains, the internal organization of labeled loci displayed less electron-dense structure. Next, we analyzed post-replication chromatin reorganization. It was found that the initial tightly packed chromonema structure was restored roughly 30 min after labeling. Experiments with PCNA and EdU double-labeling demonstrated that this restoration of chromatin structure occurs already after replication is finished. Extensive spatial segregation of EdU label and PCNA-positive replication sites was observed at this time-point by immunofluorescence. Discussion and Conclusions: The data obtained contradict with popular hypothesis of complete chromatin structure decompactization of replicating domains down to nucleosome fibers. On the other hand, we did not observed labeled replication sites surrounded by unlabeled chromatin, which questions the model of replicating chromatin organized into proteinaceous “replicative factories” with DNA spooled through clustered polymerases during replication. In contrast, we suggest that replication of higher order chromatin domains occurs without their complete decondensation, although local changes in DNA packing do take place to facilitate replication fork progression.