Authors: Laurence Vernis; Jure Piskur¹; John F. X. Diffley²

Source: Nucleic Acids Research, Volume 31, Number 19, 01 October 2003 , pp. e120-e120(1)

Publisher: Oxford University Press

Abstract:

The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse–chase labelling.

Document Type: Research article

DOI: http://dx.doi.org/10.1093/nar/gng121

Affiliations: 1: BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark 2: *To whom correspondence should be addressed. john.diffley@cancer.org.uk, Present address:, Laurence Vernis, Institut Curie UMR 2027, Université Paris Sud. Batiment 110, 91405, Orsay cedex, France, Tel: +44 20 7269 3869, Fax: +44 20 7269 3801

Publication date: 2003-10-01

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• Nucleic Acids Research (NAR) is a fully Open Access journal, providing rapid publication of leading edge research into the nucleic acids under the following categories: chemistry, computational biology, genomics, molecular biology, nucleic acid enzymes, RNA and structural biology. There is a Survey and Summary section, and methods papers are published
  in NAR Methods Online. Each year the first issue is devoted to biological databases, and a later issue to relevant web-based software resources.

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