ABSTRACT
It is considered that sister chromatids are held together immediately after replication by special protein complex--cohesin that consists of Smc1--Smc3 core dimer and two additional subunits, Scc1 and Scc3. This process is called cohesion. We have characterized binding of cohesin complex to early- and late-replicated chromatin at different stages of the cell cycle in human cells HeLa and HT1080 using superresolution microscopy (based on Structural ilumination microscopy--SIM) and immunoelectron microscopy. It has been shown that cohesins do not play important role in cohesion of heterochromatic domains, but they provide cohesion and organization of subdomains in euchromatic regions.
Subject(s)
Cell Cycle Proteins/chemistry , Chondroitin Sulfate Proteoglycans/chemistry , Chromatids/metabolism , Chromosomal Proteins, Non-Histone/chemistry , Euchromatin/metabolism , Heterochromatin/metabolism , Nuclear Proteins/chemistry , Phosphoproteins/chemistry , Cell Cycle/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Chondroitin Sulfate Proteoglycans/genetics , Chondroitin Sulfate Proteoglycans/metabolism , Chromatids/ultrastructure , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA-Binding Proteins , Euchromatin/ultrastructure , Gene Expression , HeLa Cells , Heterochromatin/ultrastructure , Humans , Microscopy, Immunoelectron , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Protein MultimerizationABSTRACT
Tight association of peripheral chromatin with nuclear lamina unavoidably creates topological constraints during replication. Additional complications are associated with high stability of lamina meshwork, which may hinder an access of replication factors to the sites of DNA synthesis in highly condensed template with limited mobility. In the current work we studied structural organization and dynamics of lamina as a function of replicative status of associated peripheral heterochromatin. The studies of molecular mobility of laminas at various stages of S-phase in vivo and using super-resolution microscopy showed no correlation between lamina dynamics and replicative status of attached heterochromatin. These data support the hypothesis that lamina-chromatin interactions during S-phase are regulated at the level of adapter proteins. Ultrastructural studies have demonstrated that temporal break of lamina-chromatin connections during replication does not cause noticeable spatial separation of replicating domains from nuclear periphery.
Subject(s)
DNA Replication , DNA/metabolism , Fibroblasts/metabolism , Heterochromatin/metabolism , Nuclear Lamina/metabolism , Animals , CHO Cells , Cell Line, Tumor , Cricetulus , Fibroblasts/cytology , Gene Expression , Heterochromatin/ultrastructure , Humans , Lamin Type A/genetics , Lamin Type A/metabolism , Lamin Type B/genetics , Lamin Type B/metabolism , Nuclear Lamina/ultrastructureABSTRACT
The non-coding and repetitive sequences constitute a great amount of higher eukaryotes genomes, but the elucidation of its role and mechanisms of action is now at the very beginning. Here we found, that internal telomeric repeats in Danio rerio are colocalized with some repetitive elements, namely, hAT and EnSpm repeats, which are highly represented in vertebrate genome. While investigating one of genome regions, containing two pairs of such repeats in close proximity we found, that it is transcribed. RNA-dependent structures, containing this sequence, were revealed in D. rerio fibroblast nuclei, which may serve as evidence of functional relevance of repetitive elements in genomes or of their transcripts.
