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

Cytocentrifugation is a common technique for the capture of cells on microscopic slides. It usually requires a special cytocentrifuge or cytorotor and cassettes. In the study presented here, we tested the new concept of cytocentrifugation based on the threaded connection of the lid and the sample holder to ensure an adjustable flow of solutions through the filters and the collection of the filtered solutions in the reservoir during centrifugation. To test this concept, we developed a device for the preparation of cell samples on circular coverslips. The device was tested for the capture and sample processing of both eukaryotic and prokaryotic cells, cell nuclei, and mitochondria for microscopy analysis including image cytometry. Moreover, an efficient procedure was developed for capturing formaldehyde-fixed cells on non-treated coverslips without cell drying. The results showed that the tested arrangement enables the effective capture and processing of all of the tested samples and the developed device represents an inexpensive alternative to common cytocentrifuges, as only the paper filter is consumed during sample processing, and no special centrifuge, cytorotor, or cassette is necessary. As no additional system of solution removal is required during sample staining, the tested concept also facilitates the eventual automation of the staining procedure.

• Keywords
• cytocentrifugation, microscopy, sample processing, staining,
• MeSH
• Staining and Labeling methods   MeSH
• Centrifugation instrumentation   methods   MeSH
• Cytological Techniques instrumentation   methods   MeSH
• Humans MeSH
• Microscopy methods   MeSH
• Specimen Handling methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article 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

The approach for the detection of replicational activity in cells using 5-bromo-2'-deoxyuridine, a low concentration of hydrochloric acid and exonuclease III is presented in the study. The described method was optimised with the aim to provide a fast and robust tool for the detection of DNA synthesis with minimal impact on the cellular structures using image and flow cytometry. The approach is based on the introduction of breaks into the DNA by the low concentration of hydrochloric acid followed by the subsequent enzymatic extension of these breaks using exonuclease III. Our data showed that the method has only a minimal effect on the tested protein localisations and is applicable both for formaldehyde- and ethanol-fixed cells. The approach partially also preserves the fluorescence of the fluorescent proteins in the HeLa cells expressing Fluorescent Ubiquitin Cell Cycle Indicator. In the case of the short labelling pulses that disabled the use of 5-ethynyl-2'-deoxyuridine because of the low specific signal, the described method provided a bright signal enabling reliable recognition of replicating cells. The optimized protocol was also successfully tested for the detection of trifluridine, the nucleoside used as an antiviral drug and in combination with tipiracil also for the treatment of some types of cancer.

• MeSH
• Bromodeoxyuridine metabolism   MeSH
• Cell Cycle * MeSH
• A549 Cells MeSH
• Exodeoxyribonucleases metabolism   MeSH
• Microscopy, Fluorescence MeSH
• HeLa Cells MeSH
• Hydrochloric Acid pharmacology   MeSH
• Humans MeSH
• Flow Cytometry MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bromodeoxyuridine MeSH
• exodeoxyribonuclease III MeSH Browser
• Exodeoxyribonucleases MeSH
• Hydrochloric Acid MeSH

5-Bromo-2'-deoxyuridine (BrdU) labelling and immunostaining is commonly used for the detection of DNA replication using specific antibodies. Previously, we found that these antibodies significantly differ in their affinity to BrdU. Our present data showed that one of the reasons for the differences in the replication signal is the speed of antibody dissociation. Whereas highly efficient antibodies created stable complexes with BrdU, the low efficiency antibodies were unstable. A substantial loss of the signal occurred within several minutes. The increase of the complex stability can be achieved by i) formaldehyde fixation or ii) a quick reaction with a secondary antibody. These steps allowed the same or even higher signal/background ratio to be reached as in the highly efficient antibodies. Based on our findings, we optimised an approach for the fully enzymatic detection of BrdU enabling the fast detection of replicational activity without a significant effect on the tested proteins or the fluorescence of the fluorescent proteins. The method was successfully applied for image and flow cytometry. The speed of the method is comparable to the approach based on 5-ethynyl-2'-deoxyuridine. Moreover, in the case of short labelling pulses, the optimised method is even more sensitive. The approach is also applicable for the detection of 5-trifluoromethyl-2'-deoxyuridine.

• MeSH
• Bromodeoxyuridine chemistry   MeSH
• Cell Cycle MeSH
• A549 Cells MeSH
• Microscopy, Fluorescence MeSH
• HeLa Cells MeSH
• Humans MeSH
• Copper chemistry   MeSH
• Antibodies chemistry   MeSH
• Flow Cytometry MeSH
• DNA Replication physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bromodeoxyuridine MeSH
• Copper MeSH
• Antibodies MeSH

5-Ethynyl-2'-deoxyuridine (EdU) and 5-ethynyl-2'-deoxycytidine (EdC) are mainly used as markers of cellular replicational activity. Although EdU is employed as a replicational marker more frequently than EdC, its cytotoxicity is commonly much higher than the toxicity of EdC. To reveal the reason of the lower cytotoxicity of EdC, we performed a DNA analysis of five EdC-treated human cell lines. Surprisingly, not a single one of the tested cell lines contained a detectable amount of EdC in their DNA. Instead, the DNA of all the cell lines contained EdU. The content of incorporated EdU differed in particular cells and EdC-related cytotoxicity was directly proportional to the content of EdU. The results of experiments with the targeted inhibition of the cytidine deaminase (CDD) and dCMP deaminase activities indicated that the dominant role in the conversion pathway of EdC to EdUTP is played by CDD in HeLa cells. Our results also showed that the deamination itself was not able to effectively prevent the conversion of EdC to EdCTP, the conversion of EdC to EdCTP occurs with much lesser effectivity than the conversion of EdU to EdUTP and the EdCTP is not effectively recognized by the replication complex as a substrate for the synthesis of nuclear DNA.

• Keywords
• 5-ethynyl-2′-deoxycytidine, 5-ethynyl-2′-deoxyuridine, DNA replication, cytidine deaminase, dCMP deaminase,
• MeSH
• Bromodeoxyuridine metabolism   MeSH
• Cell Death MeSH
• Cell Nucleus metabolism   MeSH
• Cytidine Deaminase metabolism   MeSH
• Deoxycytidine analogs & derivatives   metabolism   MeSH
• Deoxyuridine analogs & derivatives   metabolism   MeSH
• DNA metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering metabolism   MeSH
• Metabolome MeSH
• Cell Line, Tumor MeSH
• Antibodies metabolism   MeSH
• DNA Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-ethynyl-2'-deoxycytidine MeSH Browser
• 5-ethynyl-2'-deoxyuridine MeSH Browser
• Bromodeoxyuridine MeSH
• Cytidine Deaminase MeSH
• Deoxycytidine MeSH
• Deoxyuridine MeSH
• DNA MeSH
• RNA, Small Interfering MeSH
• Antibodies MeSH