scholarly journals BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin

2006 ◽  
Vol 175 (5) ◽  
pp. 693-701 ◽  
Author(s):  
Gayle J. Pageau ◽  
Jeanne B. Lawrence
Keyword(s):  
Pericentric Heterochromatin ◽  
S Phase ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Embryonic Cells ◽  
Major Satellite ◽  
Inactive X Chromosome ◽  
Xist Rna ◽  
Brdu Labeling ◽  
Proliferating Cell

Breast cancer–associated protein 1 (BRCA1) forms foci at sites of induced DNA damage, but any significance of these normal S-phase foci is unknown. BRCA1 distribution does not simply mirror or overlap that of replicating DNA; however, BRCA1 foci frequently abut sites of BrdU incorporation, mostly at mid-to-late S phase. Although BRCA1 does not overlap XIST RNA across the inactive X chromosome, BRCA1 foci position overwhelmingly in heterochromatic regions, particularly the nucleolar periphery where many centromeres reside. In humans and mice, including early embryonic cells, BRCA1 commonly associates with interphase centromere–kinetochore complexes, including pericentric heterochromatin. Proliferating cell nuclear antigen or BrdU labeling demonstrates that BRCA1 localizes adjacent to, or “paints,” major satellite blocks as chromocenters replicate, where topoisomerase is also enriched. BRCA1 loss is often associated with proliferative defects, including postmitotic bridges enriched with satellite DNA. These findings implicate BRCA1 in replication-linked maintenance of centric/pericentric heterochromatin and suggest a novel means whereby BRCA1 loss may contribute to genomic instability and cancer.

Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 389-399 ◽  
Author(s):  
E.J. Sanders ◽  
M. Varedi ◽  
A.S. French
Keyword(s):  
Cell Proliferation ◽  
Chick Embryo ◽  
Cell Density ◽  
Generation Time ◽  
Primitive Streak ◽  
S Phase ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Similar Proportion ◽  
Differential Growth

Cell proliferation in the gastrulating chick embryo was assessed using two independent techniques which mark cells in S phase of the mitotic cycle: nuclear incorporation of bromodeoxyuridine (BrdU) detected immunocytochemically and immunolocalization of proliferating cell nuclear antigen (PCNA). Computer-reconstructed maps were produced showing the distribution of labelled nuclei in the primitive streak and the cell layers. These distributions were also normalized to take into account regional differences in cell density across the embryo. Results from a 2 hour pulse of BrdU indicated that although cells at caudal levels of the primitive streak showed the highest incorporation, this region showed a similar proportion of labelled cells to the surrounding caudal regions of the epiblast and mesoderm when normalized for cell density. The entire caudal third of the embryo showed the highest proportion of cells in S phase. Cells of Hensen's node showed a relatively low rate of incorporation and, although the chordamesoderm cells showed many labelled nuclei, this appeared to be a reflection of a high cell density in this region. Combining this result with results from a 4 hour pulse of BrdU permitted mapping of cell generation time across the entire embryo. Generation times ranged from a low value of approximately 2 hours at caudal levels of both the epiblast and mesoderm, to an upper value of approximately 10 hours in the rostral regions of the primitive streak, in the mid-lateral levels of the epiblast and in the chordamesoderm rostral to Hensen's node. Cells at caudal regions of the primitive streak showed a generation time of approximately 5 hours. Taking into account that cells are generally considered to be continuously moving through the primitive streak, we conclude that cell division, as judged by generation time, is greatly reduced during transit through this region, despite the presence there of cells in S phase and M phase. Immunocytochemical localization of PCNA-positive nuclei gave generally similar distributions to those obtained with BrdU incorporation, confirming that this endogenous molecule is a useful S-phase marker during early embryogenesis. Mid-levels and caudal levels of the primitive streak showed the highest numbers of positive nuclei, and the highest proportion of labelling after cell density was accounted for. As with BrdU incorporation, the highest proportions of PCNA-positive nuclei were found towards the caudal regions of the epiblast and mesoderm. These results suggest that the differential growth of the caudal region of the embryo at this time is a direct consequence of elevated levels of cell proliferation in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals BRCa1 participates in XIST RNa localization on inactive x chromosome

2019 ◽  
Vol 18 (2) ◽  
pp. 21-26
Author(s):  
E. A. Shestakova ◽  
T. A. Bogush
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Confocal Microscopy ◽  
X Chromosome ◽  
Fluorescent Microscopy ◽  
S Phase ◽  
Immunofluorescent Staining ◽  
Heterochromatin Protein ◽  
Inactive X Chromosome ◽  
Xist Rna

Introduction . Inactive X chromosome (Xi) is associated with noncoding XIST RNA, series of proteins and contains multiple epigenetic modifications that altogether determine a silence of the most of X-linked genes. Recently the data were obtained that tumor suppressor BRCA1 is also associated with Xi. The purpose of this study was to reveal the colocalization of BRCA1 and XIST RNA and precise spatial organization on Xi with the high resolution of confocal microscopy.Materials and methods . The object of the study is IMR90hTERT diploid immortalized fibroblast cell line. For BRCA1 and XIST RNA colocalization analysis on Xi the method of fluorescent hybridization in situ associated with immunofluorescent cell staining (immunoFISH) and confocal microscopy were used. For BRCA1 and heterochromatin protein-1 colocalization study the method of double immunofluorescent staining and common fluorescent microscopy were applied. Results . The study using confocal fluorescent microscopy with higher resolution has demonstrated at first the colocalization of BRCA1 with XIST RNA region of Xi revealed with XIST RNA probes and with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. Altogether, the data obtained suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the regulation of XIST RNA association with Xi. Moreover, according to the results of confocal microscopy, BRCA1 also colocalizes with replicating Xi and autosomes revealed with BrdU in late S-phase of cell cycle. This indicates a possible involvement of this protein in the replication of pericentromeric repeats in cellular chromosomes. Colocalization of BRCA1 with heterochromatin protein-1α presented in pericentromeric regions of all chromosomes supports this suggestion.Conclusions . Altogether, the data obtained in this study suggest the involvement of BRCA1 in the inhibition of gene expression on Xi due to the association with noncoding inhibiting XIST RNA and in replication of heterochromatin regions. 

Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle

1994 ◽  
Vol 107 (8) ◽  
pp. 2191-2202 ◽  
Author(s):  
P. Hozak ◽  
D.A. Jackson ◽  
P.R. Cook
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
S Phase ◽  
Nuclear Bodies ◽  
Nuclear Antigen ◽  
Thin Sections ◽  
Permeabilized Cells ◽  
Ultrastructural Characterization ◽  
Replication Sites ◽  
Proliferating Cell

Sites of replication in synchronized HeLa cells were visualized by light and electron microscopy; cells were permeabilized and incubated with biotin-16-dUTP, and incorporation sites were immunolabelled. Electron microscopy of thick resinless sections from which approximately 90% chromatin had been removed showed that most DNA synthesis occurs in specific dense structures (replication factories) attached to a diffuse nucleoskeleton. These factories appear at the end of G1-phase and quickly become active; as S-phase progresses, they increase in size and decrease in number like sites of incorporation seen by light microscopy. Electron microscopy of conventional thin sections proved that these factories are a subset of nuclear bodies; they changed in the same characteristic way and contained DNA polymerase alpha and proliferating cell nuclear antigen. As replication factories can be observed and labelled in non-permeabilized cells, they cannot be aggregation artifacts. Some replication occurs outside factories at discrete sites on the diffuse skeleton; it becomes significant by mid S-phase and later becomes concentrated beneath the lamina.

scholarly journals Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases

2008 ◽  
Vol 19 (12) ◽  
pp. 5193-5202 ◽  
Author(s):  
Simone Sabbioneda ◽  
Audrey M. Gourdin ◽  
Catherine M. Green ◽  
Angelika Zotter ◽  
Giuseppina Giglia-Mari ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Proliferating Cell Nuclear Antigen ◽  
Dynamic Properties ◽  
Human Fibroblasts ◽  
Dna Polymerases ◽  
S Phase ◽  
Nuclear Antigen ◽  
Replication Foci ◽  
Y Family ◽  
Proliferating Cell

Y-family DNA polymerases carry out translesion synthesis past damaged DNA. DNA polymerases (pol) η and ι are usually uniformly distributed through the nucleus but accumulate in replication foci during S phase. DNA-damaging treatments result in an increase in S phase cells containing polymerase foci. Using photobleaching techniques, we show that polη is highly mobile in human fibroblasts. Even when localized in replication foci, it is only transiently immobilized. Although ubiquitination of proliferating cell nuclear antigen (PCNA) is not required for the localization of polη in foci, it results in an increased residence time in foci. polι is even more mobile than polη, both when uniformly distributed and when localized in foci. Kinetic modeling suggests that both polη and polι diffuse through the cell but that they are transiently immobilized for ∼150 ms, with a larger proportion of polη than polι immobilized at any time. Treatment of cells with DRAQ5, which results in temporary opening of the chromatin structure, causes a dramatic immobilization of polη but not polι. Our data are consistent with a model in which the polymerases are transiently probing the DNA/chromatin. When DNA is exposed at replication forks, the polymerase residence times increase, and this is further facilitated by the ubiquitination of PCNA.

scholarly journals The distribution pattern of proliferating cell nuclear antigen in the nuclei of Leishmania donovani

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3748-3757 ◽  
Author(s):  
Devanand Kumar ◽  
Neha Minocha ◽  
Kalpana Rajanala ◽  
Swati Saha
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Systemic Disease ◽  
S Phase ◽  
Nuclear Antigen ◽  
M Phase ◽  
Proliferating Cell

DNA replication in eukaryotes is a highly conserved process marked by the licensing of multiple origins, with pre-replication complex assembly in G1 phase, followed by the onset of replication at these origins in S phase. The two strands replicate by different mechanisms, and DNA synthesis is brought about by the activity of the replicative DNA polymerases Pol δ and Pol ϵ. Proliferating cell nuclear antigen (PCNA) augments the processivity of these polymerases by serving as a DNA sliding clamp protein. This study reports the cloning of PCNA from the protozoan Leishmania donovani, which is the causative agent of the systemic disease visceral leishmaniasis. PCNA was demonstrated to be robustly expressed in actively proliferating L. donovani promastigotes. We found that the protein was present primarily in the nucleus throughout the cell cycle, and it was found in both proliferating procyclic and metacyclic promastigotes. However, levels of expression of PCNA varied through cell cycle progression, with maximum expression evident in G1 and S phases. The subnuclear pattern of expression of PCNA differed in different stages of the cell cycle; it formed distinct subnuclear foci in S phase, while it was distributed in a more diffuse pattern in G2/M phase and post-mitotic phase cells. These subnuclear foci are the sites of active DNA replication, suggesting that replication factories exist in Leishmania, as they do in higher eukaryotes, thus opening avenues for investigating other Leishmania proteins that are involved in DNA replication as part of these replication factories.

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