Pentose phosphate pathway function affects tolerance to the G-quadruplex binder TMPyP4

Abstract

G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.

Figure 1. TMPyP4, G-quartet, and G-quadruplex structures (A) The structure of the porphyrin TMPyP4 (B) The structure of G-quartets, adapted from and an example of a G-quadruplex structure.

The sphere in the centre of the G-quartet represents a central cation.

Figure 2. Fitness of yfgΔ strains on TMPyP4 (A) Plate 10 of the genome-wide screen grown on CSM and CSM containing 100 µM TMPyP4.

Highlighted genes indicate five different deletion strains on the TMPyP4-containing plate. (B) Individual growth curves of the four repeats of the genes highlighted in (A) on CSM and CSM containing 100 µM TMPyP4. (C) The fitnesses of ∼4300 gene deletion mutants on media with or wihtout 100 µM TMPyP4. Each point on the graph represents a single deletion genotype. The dashed grey line indicates hypothetical 1∶1 growth under both conditions. The solid grey line indicates expected fitness based on a population model. Strains below this solid line display a reduced fitness on supplemented media than expected.

Figure 3. Comparison of TMPyP4 high-throughput screens.

Correlation plot of fitness differential values from two screens (correlation = 0.066). Genes in red (bottom left-hand corner) were found to increase TMPyP4-sensitivity when deleted in both experiments (FD ≤−0.5 in the initial screen and FFD ≤−0.2 in the follow-up screen). Genes in green (top right-hand corner) were found to decrease TMPyP4-sensitivity when deleted according to both experiments (FD ≥0.5 in the initial screen and FFD ≥0.25 in the follow-up screen). Blue lines denote FD thresholds.

Figure 4. The pentose phosphate pathway protects against sensitivity to TMPyP4.

(A) The pentose phosphate pathway. (B) Spot test for TMPyP4-sensitivity of pppΔ strains in the W303 background. Strains were grown to saturation in YEPD before a 5-fold serial dilution and spotting onto plates with or without 100 µM TMPyP4. Incubation was carried out at 30°C for 3 days. (C) Spot test for cdc13-1 interaction with TMPyP4. Strains were grown and spotted as in (B). Incubation was carried out at indicated temperatures for 3 days.

Figure 5. TMPyP4 and hydrogen peroxide treatment result in similar fitness changes.

(A) Correlation plot of fitness differential values from the TMPyP4 screen and H₂O₂ screen. Genes in red (bottom left-hand corner) were found to increase sensitivity to both TMPyP4 and H₂O₂ when deleted (FD ≤−0.5). Genes in green (top right-hand corner) were found to decrease sensitivity to both TMPyP4 and H₂O₂ when deleted (FD ≥0.5). Blue lines denote FD thresholds. (B) Spot test for TMPyP4 and H₂O₂ sensitivity. Strains were grown to saturation in YEPD before a 5-fold serial dilution in water and spotting onto plates with or without 100 µM TMPyP4. Incubation was carried out at 30°C for 3 days. Strains from three different genetic backgrounds (W303, S288C and BY4741) were tested, as indicated on the left hand side and Table S1 in File S1.

Figure 6. Exposure to light increases the sensitivity of yeast strains to TMPyP4.

Strains were grown to saturation in YEPD before a 5-fold serial dilution in water and spotting onto plates containing 0 µM, 5 µM, 50 µM or 100 µM TMPyP4. Plates were incubated at 30°C for 3 days in an incubator fitted with a light. Plates labelled ‘Dark’ were wrapped in reflective foil. Strains from three different genetic backgrounds (W303, S288C and BY4741) were tested, as indicated on the left and in Table S1 in File S1. A control spot test on plates lacking TMPyP4 (CSM) was conducted in the dark; however, the growth of strains did not differ from control plates kept in the light.