Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency

Abstract

Ribonucleotide reductase (RNR) is an essential iron-dependent enzyme that catalyzes deoxyribonucleotide synthesis in eukaryotes. Living organisms have developed multiple strategies to tightly modulate RNR function to avoid inadequate or unbalanced deoxyribonucleotide pools that cause DNA damage and genome instability. Yeast cells activate RNR in response to genotoxic stress and iron deficiency by facilitating redistribution of its small heterodimeric subunit Rnr2-Rnr4 from the nucleus to the cytoplasm, where it forms an active holoenzyme with large Rnr1 subunit. Dif1 protein inhibits RNR by promoting nuclear import of Rnr2-Rnr4. Upon DNA damage, Dif1 phosphorylation by the Dun1 checkpoint kinase and its subsequent degradation enhances RNR function. In this report, we demonstrate that Dun1 kinase triggers Rnr2-Rnr4 redistribution to the cytoplasm in response to iron deficiency. We show that Rnr2-Rnr4 relocalization by low iron requires Dun1 kinase activity and phosphorylation site Thr-380 in the Dun1 activation loop, but not the Dun1 forkhead-associated domain. By using different Dif1 mutant proteins, we uncover that Dun1 phosphorylates Dif1 Ser-104 and Thr-105 residues upon iron scarcity. We observe that the Dif1 phosphorylation pattern differs depending on the stimuli, which suggests different Dun1 activating pathways. Importantly, the Dif1-S104A/T105A mutant exhibits defects in nucleus-to-cytoplasm redistribution of Rnr2-Rnr4 by iron limitation. Taken together, these results reveal that, in response to iron starvation, Dun1 kinase phosphorylates Dif1 to stimulate Rnr2-Rnr4 relocalization to the cytoplasm and promote RNR function. Ribonucleotide reductase (RNR)5 catalyzes the rate-limiting step in the de novo deoxyribonucleotide (dNTP) synthesis by converting ribonucleoside diphosphates to the corresponding deoxy forms. In eukaryotes, the RNR holoenzyme is composed of a large or R1 subunit that contains the catalytic and allosteric sites, and a small or R2 subunit that harbors a di-iron center, which is responsible for generating and keeping a tyrosyl radical required for catalysis (reviewed in Refs.

Expression of mammalian Onzin and Fungal Cadmium Resistance 1 in S. cerevisiae suggests ancestral functions of PLAC8 proteins in regulating mitochondrial metabolism and DNA damage repair

“…Abbà and colleagues [19] found that Dun1p was necessary for the cadmium tolerant phenotype of FCR1 in a Mec1p-independent pathway. Here, we show that Dun1p was also required for the cadmium tolerant phenotype of Onzin-expressing yeast (Fig 5), suggesting that both PLAC8 proteins may activate a specific Dun1p-dependent DNA damage checkpoint pathway similar to the one described by Pijuan et al, [62] and Sanvisens et al, [63]. Indeed, Dun1p activates the RNR at multiple levels (i.e.…”

“…By contrast, dysfunctions of the core mitochondrial ISC assembly machinery induced a second pathway involving a Mec1p-independent activation of Dun1p. Similarly, Sanvisens and colleagues [63] found that depletion of core components of the mitochondrial ISC assembly activated a Dun1p-dependent but Mec1p- and Rad53p-independent pathway leading to increased dNTPs biosynthesis, needed for DNA repair.…”

Iron Regulatory Mechanisms in Saccharomyces cerevisiae

“…In fact, the levels of UFAs have been reported to be crucial for the Aft1-dependent activation of the iron regulon, although no mechanistic clues have been provided yet ( Jorda et al, 2020 ). Moreover, both iron deficiency and defects in the mitochondrial ISC biogenesis activate the Mec1-Rad53-Dun1 DNA damage checkpoint kinase cascade that, in combination with Cth2 and Aft1 regulatory factors (see above), promote the activity of the iron-dependent RNR enzyme at multiple levels [( Sanvisens et al, 2011 , 2014 , 2016 ; Pijuan et al, 2015 ; Ros-Carrero et al, 2020 ); reviewed in Sanvisens et al (2013) ; Puig et al (2017) ]. Upon iron limitation, Dun1 kinase enhances dNTP synthesis by promoting the degradation of the R1 inhibitor protein Sml1, and the R2 import protein Dif1 ( Sanvisens et al, 2014 , 2016 ).…”

DNA damage response activates respiration and thereby enlarges dNTP pools to promote cell survival in budding yeast

“…into the cytoplasm, where they assemble with Rnr1p to form an active RNR enzyme (25)(26)(27)(28)(29)(30). During S phase or after DNA damage, Dun1p also phosphorylates and induces degradation of Sml1p, a protein that binds and inhibits the Rnr1p subunit ( Fig.…”