Mismatched nucleobase uracil is commonly repaired through the base excision repair initiated by DNA uracil glycosylases. The data presented in this study strongly indicate that the nuclear uracil-N-glycosylase activity and nuclear protein content in human cell lines is highest in the S phase of the cell cycle and that its distribution kinetics partially reflect the DNA replication activity in replication foci. In this respect, the data demonstrate structural changes of the replication focus related to the uracil-N-glycosylase distribution several dozens of minutes before end of its replication. The analysis also showed that very popular synchronisation protocols based on the double thymidine block can result in changes in the UNG2 content and uracil excision rate. In response, we propose a new method for the description of the changes of the content and the activity of different cell components during cell cycle without the necessity to use synchronisation protocols.

• MeSH
• Cell Cycle MeSH
• Kinetics MeSH
• Humans MeSH
• DNA Repair MeSH
• DNA Replication * MeSH
• S Phase MeSH
• Uracil-DNA Glycosidase * metabolism   MeSH
• Uracil metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Uracil-DNA Glycosidase * MeSH
• Uracil MeSH

Cellular growth and the preparation of cells for division between two successive cell divisions is called the cell cycle. The cell cycle is divided into several phases; the length of these particular cell cycle phases is an important characteristic of cell life. The progression of cells through these phases is a highly orchestrated process governed by endogenous and exogenous factors. For the elucidation of the role of these factors, including pathological aspects, various methods have been developed. Among these methods, those focused on the analysis of the duration of distinct cell cycle phases play important role. The main aim of this review is to guide the readers through the basic methods of the determination of cell cycle phases and estimation of their length, with a focus on the effectiveness and reproducibility of the described methods.

• Keywords
• BrdU, DNA labeling, EdU, cell cycle, labeled nucleosides, markers of cell cycle phases, time lapse microscopy,
• MeSH
• Bromodeoxyuridine * metabolism   MeSH
• Cell Division MeSH
• Cell Cycle MeSH
• Cell Proliferation MeSH
• Reproducibility of Results MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Bromodeoxyuridine * MeSH

Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available. In this review, the basic techniques used for cell quantification, with a special emphasis on techniques based on fluorescent DNA dyes, are described. The main aim of this review is to guide readers through the possibilities of cell quantification with various methods and to show the strengths and weaknesses of these methods, especially with respect to their sensitivity, accuracy, and length. As these methods are frequently accompanied by an analysis of cell proliferation and cell viability, some of these approaches are also described.

• Keywords
• DNA dyes, cell metabolism, cell quantification, enzymatic conversion of substrate,
• MeSH
• Biological Assay MeSH
• DNA analysis   chemistry   metabolism   MeSH
• Fluorescent Dyes * MeSH
• Cell Count methods   MeSH
• Cell Proliferation MeSH
• Cell Survival MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Comparative Study MeSH
• Names of Substances
• DNA MeSH
• Fluorescent Dyes * MeSH

The replication of nuclear and mitochondrial DNA are basic processes assuring the doubling of the genetic information of eukaryotic cells. In research of the basic principles of DNA replication, and also in the studies focused on the cell cycle, an important role is played by artificially-prepared nucleoside and nucleotide analogues that serve as markers of newly synthesized DNA. These analogues are incorporated into the DNA during DNA replication, and are subsequently visualized. Several methods are used for their detection, including the highly popular click chemistry. This review aims to provide the readers with basic information about the various possibilities of the detection of replication activity using nucleoside and nucleotide analogues, and to show the strengths and weaknesses of those different detection systems, including click chemistry for microscopic studies.

• Keywords
• click chemistry, indirect immunocytochemistry, isotopes, nucleoside and nucleotide analogues,
• MeSH
• Click Chemistry MeSH
• DNA chemistry   genetics   MeSH
• Halogenation MeSH
• In Situ Hybridization MeSH
• Immunohistochemistry MeSH
• Isotope Labeling MeSH
• Copper chemistry   MeSH
• Nucleosides chemistry   MeSH
• Nucleotides chemistry   MeSH
• Radioisotopes MeSH
• DNA Replication * MeSH
• Research MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• DNA MeSH
• Copper MeSH
• Nucleosides MeSH
• Nucleotides MeSH
• Radioisotopes MeSH

According to a general paradigm, proper DNA duplication from each replication origin is ensured by two protein complexes termed replisomes. In prokaryotes and in budding yeast Saccharomyces cerevisiae, these two replisomes seem to be associated with one another until DNA replication initiated from the origin has finished. This arrangement results in the formation of the loop of newly synthesized DNA. However, arrangement of replisomes in other eukaryotic organisms including vertebrate cells is largely unknown. Here, we used in vivo labeling of DNA segments in combination with the electron microscopy tomography to describe the organization of replisomes in human HeLa cells. The experiments were devised in order to distinguish between a model of independent replisomes and a model of replisome couples. The comparative analysis of short segments of replicons labeled in pulse-chase experiments of various length shows that replisomes in HeLa cells are organized into the couples during DNA replication. Moreover, our data enabled to suggest a new model of the organization of replicated DNA. According to this model, replisome couples produce loop with the associated arms in the form of four tightly associated 30nm fibers.

• MeSH
• Bromodeoxyuridine metabolism   MeSH
• Cell Nucleus metabolism   ultrastructure   MeSH
• Chromatin physiology   ultrastructure   MeSH
• Deoxyuracil Nucleotides metabolism   MeSH
• DNA-Directed DNA Polymerase chemistry   metabolism   MeSH
• HeLa Cells MeSH
• Nucleic Acid Conformation MeSH
• Humans MeSH
• Models, Genetic * MeSH
• Multienzyme Complexes chemistry   metabolism   MeSH
• Image Processing, Computer-Assisted MeSH
• DNA Replication physiology   MeSH
• Replicon genetics   MeSH
• Electron Microscope Tomography MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bromodeoxyuridine MeSH
• Chromatin MeSH
• Deoxyuracil Nucleotides MeSH
• DNA synthesome MeSH Browser
• DNA-Directed DNA Polymerase MeSH
• Multienzyme Complexes MeSH

Pontin is a multifunctional protein having roles in various cellular processes including regulation of gene expression. Here, we addressed Pontin intracellular localization using two different monoclonal antibodies directed against different Pontin epitopes. For the first time, Pontin was directly visualized in nucleoli where it co-localizes with Upstream Binding Factor and RNA polymerase I. Nucleolar localization of Pontin was confirmed by its detection in nucleolar extracts and by electron microscopy, which revealed Pontin accumulation specifically in the nucleolar fibrillar centers. Pontin localization in the nucleolus was dynamic and Pontin accumulated in large nucleolar dots mainly during S-phase. Pontin concentration in the large nucleolar dots correlated with reduced transcriptional activity of nucleoli. In addition, Pontin was found to associate with RNA polymerase I and to interact in a complex with c-Myc with rDNA sequences indicating that Pontin is involved in the c-Myc-dependent regulation of rRNA synthesis.

• MeSH
• ATPases Associated with Diverse Cellular Activities MeSH
• Cell Nucleolus enzymology   ultrastructure   MeSH
• DNA Helicases metabolism   MeSH
• Transcription, Genetic MeSH
• HeLa Cells MeSH
• Humans MeSH
• RNA, Ribosomal biosynthesis   MeSH
• RNA Polymerase I metabolism   MeSH
• Pol1 Transcription Initiation Complex Proteins metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Carrier Proteins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ATPases Associated with Diverse Cellular Activities MeSH
• DNA Helicases MeSH
• RNA, Ribosomal MeSH
• RNA Polymerase I MeSH
• RUVBL1 protein, human MeSH Browser
• transcription factor UBF MeSH Browser
• Pol1 Transcription Initiation Complex Proteins MeSH
• Carrier Proteins MeSH