Antifolate response in replication arrest mutants of Saccharomyces cerevisiae.

Aim: Thymidine deprivation is a common cancer treatment. This study examines the role of replication arrest and uracil DNA repair in response to thymidine deprivation. Materials and Methods: Strains of S. cerevisiae deficient in various replication and DNA repair functions were tested for sensitivity to thymidine deprivation induced by the antifolate aminopterin. Cell survival and DNA content were assayed following drug treatment. Results: Most arrest mutants were more sensitive to aminopterin than was the parental strain. Inactivation of uracil glycosylase in arrest mutants led to a partial reduction in toxicity for some double-mutants. DNA content during exposure to aminopterin was similar in parental and single mutants. However, cells deficient in both arrest and uracil glycosylase functions exhibited continued DNA synthesis, suggesting that uracil glycosylase activity contributes to replication arrest during thymidine deprivation. Conclusion: Replication arrest and uracil DNA repair are important and overlapping determinants of cellular response to thymidine deprivation. Nucleotide pool limitations incite a number of responses in Saccharomyces cerevisiae. Depletion of deoxyribonucleotides (dNTPs), either by treatment with hydroxyurea or ribonucleotide reductase inactivation, disrupts replication resulting in S-phase arrest (1). DNA is structurally sensitive during replication arrest. It is unwound, partially single- stranded and significantly more vulnerable to damaging agents during S-phase arrest (2, 3). A number of proteins are thought to be active in sensing and responding to replication stress caused by nucleotide pool depletion. These proteins serve to recognize replication difficulties or DNA damage, transmit appropriate signals and stabilize DNA. The roles of several proteins have been described during nucleotide pool depletion caused by decreased ribonucleotide reductase activity, which reduces the abundance of all four dNTPs. However, the role of these proteins in responding to selective depletion of thymidine has not been fully-elucidated. Thymidine deprivation poses unique challenges. Thymidine monophosphate (dTMP) is produced by thymidylate synthase in a reaction that transfers a one-carbon group from reduced folate to dUMP to produce dTMP. Anti-folates block this reaction, leading to elevated levels of the precursor dUMP and subsequently of dUTP (4, 5). dUTP and dTTP are chemically similar and most DNA polymerases have poor discrimination between dUTP and dTTP. Therefore, when thymidylate synthase is inhibited, the dUTP level rises, the dTTP level drops and these conditions favor incorporation of dUTP into DNA (5, 6). Once incorporated into DNA, uracil becomes a target for uracil glycosylase DNA base repair, which involves base excision and strand incision. These activities could be harmful if performed on DNA already in vulnerable conformations due to stalled replication. How do cells balance these potentially conflicting processes of cell-cycle arrest and uracil base excision repair? Understanding how arrest and repair activities are coordinated during thymidine deprivation may provide with unique insights into how these pathways interact. This study examines the role of replication arrest during thymidine deprivation. Interactions between arrest and uracil base excision repair are also examined.