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.
Highlights
TMPyP4 (5,10,15,20-tetratkis-(N-methyl-4-pyridyl)-21,23-Hporphyrin) (Figure 1a) is a widely used G-quadruplex binding molecule
We found no evidence of an over-representation of telomere associated genes amongst the strains found to be most sensitive to TMPyP4, instead observing that genes associated with the pentose phosphate pathway (PPP), the oxidative stress response and tubulin folding demonstrated highest TMPyP4-sensitivity upon deletion
There may exist a link between the oxidative phase of the PPP and the DNA damage response (DDR), through modulation of glucose-6-phosphate dehydrogenase activity by the DDR effector ATM [46]
Summary
TMPyP4 (5,10,15,20-tetratkis-(N-methyl-4-pyridyl)-21,23-Hporphyrin) (Figure 1a) is a widely used G-quadruplex binding molecule. The interaction between the porphyrin TMPyP4 and nucleic acid structures which form in guanine-rich regions of DNA and RNA, known as G-quadruplexes (Figure 1b), has been studied extensively [1,2,3,4]. It has been observed that TMPyP4 has the ability to inhibit telomerase activity in vitro and affect the c-MYC oncogene-dependent transcription of several genes in HeLa cells, including TERT, which encodes the human telomerase subunit [9,10,11]. This interaction with c-MYC suggests that the promoter region has G-quadruplex forming potential [12]. The formation of Gquadruplexes in vivo is undetermined, and the capability of TMPyP4 to bind G-quadruplexes in vivo is unknown [16]
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