Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae.
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.
D001973:Bromodeoxyuridine / Q000378:metabolism; D002455:Cell Division; D004271:DNA, Fungal / Q000378:metabolism; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000235:genetics
D004271:DNA, Fungal; D033721:Equilibrative Nucleoside Transporter 1; C438649:SLC29A1 protein, human; D013940:Thymidylate Synthase; D013937:Thymidine Kinase; D001973:Bromodeoxyuridine; D013936:Thymidine