Human cell free extracts are capable of carrying out damage-induced DNA synthesis in response to DNA damage by UV, psoralen, and cisplatin. We show that this damage-induced DNA synthesis is associated with removal of psoralen adducts and therefore is 'repair synthesis' and not an aberrant DNA synthesis reaction potentiated by DNA deformed by adducts. By comparing the denaturable fraction of psoralen adducted DNA which becomes labeled in the repair reaction to that of terminally labeled DNA (without repair) we have found that all DNA synthesis induced by psoralen monoadducts is the consequence of removal of these adducts. By the same approach we have obtained preliminary evidence that this in vitro system is capable of removing psoralen crosslinks as well.

Strains of Saccharomyces cerevisiae termed sphingolipid compensatory (SLC) do not grow at low pH when the cells lack sphingolipids. To begin to understand why sphingolipids are required for growth at low pH, we isolated derivatives of SLC strains, termed low pH resistant (LprR), carrying the LPR suppressor gene that allows growth at pH 4.1 when cells lack sphingolipids. Suppression is due to mutation of a single nuclear gene. The LPR suppressor gene functions, at least in part, by enhancing the ability of cells lacking sphingolipids to generate a net efflux of protons in suspension fluid with a pH range of 4.0-6.0. The LPR suppressor gene also enables cells lacking sphingolipids to maintain their intracellular pH near neutrality when the pH of the suspension fluid is low, unlike cells lacking the suppressor gene, which cannot maintain their intracellular pH in the face of a low external pH. These results demonstrate that some functions(s) of sphingolipids necessary for growth at low pH can be bypassed by a suppressor mutation. Attempts to clone the LPR suppressor gene were not successful, but they led to the isolation of the CWP2 gene, which encodes a major mannoprotein component of the outer cell wall. It was isolated because an increased copy number has the unusual property of increasing the frequency at which LprR strains arise. As we show here, part of the reason for this effect is that the CWP2 gene is essential for generating a net efflux of protons and for controlling intracellular pH in LprR strains that lack sphingolipids. These results suggest new cellular functions for the Cwp2 protein.

Cancer stem cells and mutant p53 expressing tumors are highly dependent on the mevalonate pathway. Statins inhibit this pathway resulting in growth inhibition of these cells independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Protein prenylation substrates farnesyl‐ and geranylgeranyl diphosphates are synthesized by the mevalonate pathway without producing their corresponding isoprenols. However, cells incorporate exogenously supplied isoprenols into mevalonate pathway metabolites and proteins using a poorly characterized process. In order to quantify this alternative to the mevalonate pathway, we synthesized natural and unnatural analogs of farnesol and geranylgeraniol, including stable isotope‐labeled farnesol and aniline substituted isoprenols. Using tandem mass spectrometry we quantitatively monitored the formation of diphosphate derivatives of these isoprenols and their utilization as prenylation substrates when incubated with cultured cells. Our results reveal that in some cells the capacity to use exogenous isoprenols is comparable to that of the mevalonate pathway raising the possibility that this alternative pathway could have important therapeutic implications