Metabolic remodeling maintains a reducing environment for rapid activation of the yeast DNA replication checkpoint

Abstract

Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes.

A DNA damage–induced phosphorylation circuit enhances Mec1 ATR Ddc2 ATRIP recruitment to Replication Protein A

“…In the cell, zinc is tightly bound in metal–protein complexes with limited labile pools of exchangeable ions. However, the overall available concentration of zinc more than exceeds the Kd values measured in our study, suggesting it is plausible that transient increases in cellular zinc, or zinc release through reductive stress ( 54 , 55 ), may play a physiological role in the Mec1-checkpoint.…”

A DNA damage-induced phosphorylation circuit enhances Mec1ATR-Ddc2ATRIPrecruitment to Replication Protein A

“…In the cell, zinc is tightly bound in metal-protein complexes with limited labile pools of exchangeable ions. However, the overall available concentration of zinc more than exceeds the Kd values measured in our study, suggesting it is plausible that transient increases in cellular zinc, or zinc release through reductive stress (Li et al, 2022; Manford et al, 2021), may play a physiological role in the Mec1-checkpoint.…”

Modification of Phosphorylation Sites in the Yeast Lysine Methyltransferase Set5 Exerts Influences on the Mitogen-Activated Protein Kinase Hog1 under Prolonged Acetic Acid Stress

“…These results also suggest dynamic regulation of the central protein kinase Hog1 under different environmental conditions. The phosphomimetic and alanine substitution mutations are widely used to study protein phosphorylation ( 37 , 38 ). However, no studies have been performed to test whether such modifications affect protein folding, stability, or expression levels.…”

“…Our results provide clues for further exploration of Set5 phosphorylation in the kinase-mediated stress signaling network. The substitutions we employed are commonly used to study protein phosphorylation ( 37 , 38 ). However, we cannot rule out the possibility that the mutations may affect protein folding and/or stability of Set5, which deserves further investigation.…”