Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways

Abstract

The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In Saccharomyces cerevisiae, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1-4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the RNR genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits. E ukaryotic cells have evolved complex surveillance mechanisms (i.e., checkpoints) to respond to genotoxic stress by arresting the cell cycle and inducing the transcription of genes that facilitate repair (1, 2). Failure of DNA damage response can result in genomic instability and cancer predisposition (3, 4). In mammalian cells the protein kinases ATM, ATR, and CHK2 are crucial for activating signaling pathways for cell survival after DNA damage (5-7). In the yeast Saccharomyces cerevisiae, the ATR homologue Mec1 and CHK2 homologue Rad53 are key regulators of cellular response to DNA damage, controlling the G 1 , S, and G 2 cell cycle checkpoints as well as transcriptional induction (8). Dun1, a protein kinase similar to Rad53, is also involved in these processes (9, 10). Among the best-studied transcriptional targets of the Mec1͞ Rad53͞Dun1 checkpoint pathway are the genes encoding ribonucleotide reductase (RNR; refs. 9 and 11-13).The enzymatic activity of RNR depends on the formation of a complex between two different subunits, R1 and R2. The large subunit R1 is a dimer and contains the active site for reduction of nucleoside diphosphate (NDP) substrates and the effector sites that control substrate specificity and enzymatic activity. The small subunit R2 is also a dimer that houses the di-iron tyrosyl radical (Y⅐) cofactor essential for NDP reduction. The active form of RNR is proposed to be a 1:1 complex of R1 and R2 (14-16).In budding yeast there are four RNR genes, two that code for a large subunit (RNR1 and RNR3) and two that code for a s...

Determination of the in Vivo Stoichiometry of Tyrosyl Radical per ββ‘ in Saccharomyces cerevisiae Ribonucleotide Reductase

“…MHY343 cells subjected to genotoxic stress induced by 50 mM HU show no obvious growth defect relative to the wt strain on solid YPD medium (data no shown). Finally, FLAG β shows the same nucleus to cytoplasm shuttling seen with wt β in response to S-phase or genotoxic stress (M. Huang, unpublished results, and ref 46). Thus, the presence of the 10-amino acid tag does not grossly affect β function in vivo.…”

“…The common features of RNR regulation are the allosteric effectors that control, in part, the activity and substrate specificity and transcription factors that control the mRNA levels of the subunits as a function of the cell cycle (24,(63)(64)(65)(66). In eukaryotic systems, a third common regulatory mechanism involves sequestration of the subunits in different compartments within the cell (46,67,68). In many other organisms, specific regulatory mechanisms have also been described.…”

Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

“…Yeast ribonucleotide reductase, a multi-protein complex, catalyzes one step in the dNTP production pathway. It is activated both as a normal part of the cell cycle and also in response to DNA damage [21,22,23,24,25]. RNR1 mRNA levels vary considerably with the cell cycle; the peak level occurs in the G1 phase [26].…”

Ccr4 contributes to tolerance of replication stress through control ofCRT1mRNA poly(A) tail length

“…Crt1-repressed targets include the RNR2, RNR3 and RNR4 genes that encode the DNA-damageinducible subunits of RNR, CRT1 itself, as well as HUG1, which encodes a small protein that negatively regulates MEC1-dependent checkpoint responses (Basrai et al, 1999). Finally, Rnr2 and Rnr4 redistribute from the nucleus to the cytoplasm after DNA damage in a Mec1-Rad53-Dun1-dependent manner (Yao et al, 2003).…”