MECHANISM OF THYMINELESS DEATH: 73

Abstract

As a model for thymidylate deficiency we have studied cultured mammalian cells treated with methotrexate in the presence of a purine supplement (Hx). Associated with the fall in intracellular dTTP, there is greater than 1000-fold increase in dUTP, which is normally undetectable (<0.3 nM) (due to the normal dUTPase mechanism) but under these circumstances approaches, and can even exceed, dTTP. Since DNA polymerases do not distinguish between dUTP and dTTP, dUMP is incorporated into DNA, the first demonstration of uracil in eukaryotic DNA. Uracil is rapidly removed from the DNA by uracil-DNA glycosylase followed by excision repair of the resulting apyrimidinic sites. However, because of the high dUTP/dTTP ratio, repair results in re-insertion of uracil, leading to a futile cycle of removal and reinsertion, with the possibility (especially when dUTP exceeds dTTP) of progressively widening single-stranded gaps in the DNA. We have proposed that these hitherto unsuspected lesions may contribute to the toxicity of thymidylate deprivation. We have now been able to show that when dUTP is increased in cells, even in the presence of normal dTTP (produced by treating cells with dUrd in HAT medium), newly synthesized DNA is fragmented and the cells die, as with limiting thymidylate. Finally, the toxicity of low dTTP (caused by treating cells with methotrexate and Hx) is mitigated when dUTP is lowered (with glucosamine). These results strongly support an important role for elevated dUTP in thymineless death, through a mechanism of DNA damage resulting from uracil insertion and removal.

Log in or create a free account to read this content

Gain free access to this article, as well as selected content from this journal and more on nature.com

Access through your institution

or

Sign in or create an account

Continue with Google

Continue with ORCiD