AP endonuclease deficiency results in extreme sensitivity to thymidine deprivation.
Thymidine depletion is toxic to virtually all actively growing cells. The fundamental mechanism responsible for thymidineless death remains unknown. One event thought to be critical in causing the toxicity of thymidine depletion is a sharp rise in the ratio of dUTP to dTTP and subsequent incorporation of dUTP into DNA. Maneuvers to alter dUTP levels appear to alter the toxicity of thymidine depletion. However, loss of uracil-DNA-N-glycosylase activity does not appear to change the toxicity of thymidine deprivation significantly. This study proposes to define the role of uracil base excision repair (BER) in mediating thymidineless death. The toxicity of thymidine deprivation induced by the antifolate aminopterin was measured in a series of mutant Saccharomyces cerevisiae strains deficient in various steps in uracil-BER. Most mutants displayed modest changes in their sensitivity to aminopterin, with the exception of cells lacking the abasic endonuclease Apn1. apn1 mutants displayed a profound sensitivity to aminopterin that was relieved in an apn1 ung1 double mutant. Wild-type and apn1 mutants displayed similar levels of DNA damage and S-phase arrest during aminopterin treatment. A significant portion of cell killing occurred after removal of aminopterin in both wild-type and apn1 mutant cells. apn1 mutants showed a complete inability to re-initiate DNA replication following removal of aminopterin. These findings suggest recovery from arrest is a crucial step in determining the response to thymidine deprivation and that interruptions in uracil-BER increase the toxicity of thymidine deprivation by blocking re-initiation of replication rather than inciting global DNA damage. Inhibition of apurinic/apyrimidinic endonuclease may therefore be a reasonable approach to increase the efficacy of anticancer chemotherapies based on thymidine depletion.
Dornfeld
Ken
K
Department of Radiation Oncology, University of Iowa Health Care, 200 Hawkins Drive, Iowa City, IA 52242, USA. kenneth-dornfeld@uiowa.edu
Johnson
Monika
M
D000630:Aminopterin / Q000633:toxicity; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000502:physiology*; D005493:Folic Acid Antagonists / Q000633:toxicity; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D013936:Thymidine / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism
D005493:Folic Acid Antagonists; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; C083274:Apn1 protein, S cerevisiae; D045643:DNA Repair Enzymes; D000630:Aminopterin; D014544:Uridine Triphosphate; D013936:Thymidine