ИСТИНА

Chromatin of mammalian cells is associated with the nuclear envelope through hundreds of discrete long lamina-associated domains representing approximately 35%–40% of the mammalian. It is known that the peripheral chromatin is firmly attached to the lamina. This attachment creates certain topological problems during DNA replication in peripheral chromatin that occurs in mid-to-late S-phase. These problems may be solved by transient dissociation of chromatin from nuclear lamina, the process that requires precise spatio-temporal coordination with DNA replication. This question is important not only for understanding the physiology of heterochromatin, but also the spatial organization of DNA replication in the cell. Materials and methods: HeLa cells were grown in Dulbecco’s modified Eagle’s medium containing 2 mM L-glutamine, 2 mM penicillin/streptomycin, and 10% fetal calf serum. Newly synthesized chromatin was labeled by adding the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) to the cell culture medium at a final concentration of 10mM for 15 min. Detection of EdU for fluorescent and electron microscopy was based on a click reaction with the azide-containing Alexa-488 fluorochrome in order to preserve native chromatin structure. For electron microscopy the label was visualized with anti-Alexa-488 antibodies and secondary detection with 1.4.nm Nanogold-coupled antibodies with subsequent Ag-amplification. Results: Investigation of the spatial organization of the peripheral chromatin replication revealed that the label in replicating domains was distributed irregularly, being predominantly concentrated on the surface of the domains. Two types of replicating structures were identified. In case the replication occurred on the side facing lamina, chromatin locally detached from the nuclear envelope. If replication proceeded on the side facing toward the center of the nucleus, detachment from the nuclear envelope was not observed. Furthermore, the replication of heterochromatin didn't correlate with the significant chromatin decondensation of higher-order fibrils, only a partial decrease of the chromatin density in the labeled domains was documented. Discussion and Conclusions: The difference of the spatial organization of lamina-attached chromatin replication could be based on some reasons. It could reflect the differences in the functional state of chromatin or its underlying nuclear envelope. Also, facultative and constitutive heterochromatin duplication may be using different mechanism of replication. The label distribution suggested that replication occurs on the surface of the compacted chromatin fibrils. It makes the local detachment of chromatin necessary for the access of replication factors for DNA synthesis. However, we cannot exclude the possibility that the distribution of label on the surface is caused by low availability of the antigen within the compact chromatin and does not elucidate a complete picture of replication.