Author/Editor		Vernis, Laurence; Piškur, Jure; Diffley, John FX
Title		Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae
Type		članek
Source		Nucleic Acids Res
Vol. and No.		Letnik 31, št. 19
Publication year		2003
Volume		str. e120
Language		eng
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.
Descriptors		BROMODEOXYURIDINE CELL DIVISION DNA, FUNGAL GENE DELETION GENE EXPRESSION S PHASE SACCHAROMYCES CEREVISIAE SIMPLEXVIRUS THYMIDINE THYMIDINE KINASE