Cell-cycle analyses using thymidine analogues in fission yeast

PLoS One. 2014 Feb 13;9(2):e88629. doi: 10.1371/journal.pone.0088629. eCollection 2014.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bromodeoxyuridine / metabolism
  • Bromodeoxyuridine / pharmacology
  • Cell Cycle / drug effects*
  • Cell Proliferation / drug effects
  • DNA Replication / drug effects*
  • DNA, Fungal / biosynthesis*
  • Deoxyuridine / analogs & derivatives
  • Deoxyuridine / metabolism
  • Deoxyuridine / pharmacology
  • Schizosaccharomyces / drug effects*
  • Schizosaccharomyces / growth & development
  • Schizosaccharomyces / metabolism
  • Staining and Labeling / methods
  • Thymidine / analogs & derivatives
  • Thymidine / metabolism
  • Thymidine / pharmacology*

Substances

  • DNA, Fungal
  • 5-chloro-2'-deoxyuridine
  • Bromodeoxyuridine
  • 5-ethynyl-2'-deoxyuridine
  • Thymidine
  • Deoxyuridine

Grants and funding

This work was supported by the Research Council of Norway, the Norwegian Cancer Society, the South-Eastern Norway Regional Health Authority and the Radium Hospital Legacy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.