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.
Highlights
Understanding the mechanisms of cell-cycle regulation and the maintenance of genomic integrity is a major objective of cancer research
In this work we have studied these parameters in the fission yeast Schizosaccharomyces pombe
In this study we have improved and refined the use of thymidine analogues to allow their detectable labelling in fission yeast cells with a minimum of cell-cycle perturbation
Summary
Understanding the mechanisms of cell-cycle regulation and the maintenance of genomic integrity is a major objective of cancer research. A powerful tool for monitoring and quantifying DNA replication, repair and recombination is to label the DNA with nucleoside analogues [1,2,3,4,5,6,7] Examples of such analogues are 5-bromo-29-deoxyuridine (BrdU), 5-Chloro-29deoxyuridine (CldU), 5-Iodo-29-deoxyuridine (IdU), and 5-ethynyl-29-deoxyuridine (EdU). There are two independent strains available carrying the hENT1 transporter and thymidine kinase (Table 1); one constructed by the Rhind lab [7] and another one constructed by the Forsburg lab [4] Using these strains, the DNA has been successfully labelled with BrdU, CldU, IdU and EdU [3,4,5,7,15]. In this study we have improved and refined the use of thymidine analogues to allow their detectable labelling in fission yeast cells with a minimum of cell-cycle perturbation. We have addressed which analogue is best for cell-cycle analyses, how sensitive the method is and how to double-label the DNA with two different analogues
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