Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast

Abstract

Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2′-deoxyuridine (EdU) and 5-Chloro-2′-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2′-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry.

Visualizing nucleic acid metabolism using non-natural nucleosides and nucleotide analogs

“…The labeling efficiencies of EdU versus halogentated pyrimidines have also been extensively studied at the cellular level using fission yeast as the model organism (83). In this study, Anda et al demonstrated that both nucleoside analogs adversely affect cell cycle progression (83). However, these effects can be minimized by modifying the concentrations of each analog.…”

“…However, these effects can be minimized by modifying the concentrations of each analog. In addition, applying pulse-chase techniques allowed for the sequential labelling of two consecutive S-phases of the cell cycle (83). Results from this study demonstrated that using these analogs to "double label" nucleic acid during replicative DNA synthesis provides a much more sensitive method to measure cell-cycle progression compared to conventional flow cytometry approaches employing propidium iodide as a marker for DNA content (83).…”

Evaluating the Genotoxic and Cytotoxic Effects of Thymidine Analogs, 5-Ethynyl-2′-Deoxyuridine and 5-Bromo-2′-Deoxyurdine to Mammalian Cells

“…Among them, the bromo-substituent of BrdU is closely identical to that of a methyl residue. However, halogenated pyrimidines including BrdU exhibit cytotoxic and mutagenic properties [ 17 ]. High concentrations of BrdU treatment increase SCE frequency [ 18 ] while influencing a variety of mutations [ 11 , 19 ].…”

Detecting Hematopoietic Stem Cell Proliferation Using BrdU Incorporation

“…Therefore, if DNA structure is important for other assays, it could be advantagous to use EdU staining over BrdU despite its higher cost. One potential disadvantage of both methods is that the analogs themselves can damage cells and cause mutations, making downstream, long-term experiments problematic (22, 23). Despite this caveat, these techniques are especially useful for cells types that are not highly proliferative as incorporation can be tracked in vivo over the course of several days.…”