DNA damage and cell cycle regulation of ribonucleotide reductase

Abstract

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. In addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. mRNAs for both subunits are cell cycle regulated and inducible by DNA damage in all organisms examined, including E. coli, S. cerevisiae and H. sapiens. This DNA damage regulation is thought to provide a metabolic state that facilitates DNA replicational repair processes. S. cerevisiae also encodes a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. Genetic analysis of the DNA damage response in S. cerevisiae has shown that RNR expression is under both positive and negative control. Among mutants constitutive for RNR expression are the general transcriptional repression genes, SSN6 and TUP1. Mutations in POL1 and POL3 also activate RNR expression, indicating that the DNA damage sensory network may respond directly to blocks in DNA synthesis. A protein kinase, Dun1, has been identified that controls inducibility of RNR1, RNR2 and RNR3 in response to DNA damage and replication blocks. This result suggests that the RNR genes in S. cerevisiae form a regulon that is coordinately regulated by protein phosphorylation in response to DNA damage.

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].…”

Checkpoint deficient rad53-11 yeast cannot accumulate dNTPs in response to DNA damage

“…Normal transcriptional regulation of RNR is governed by a cell-cycle-dependent mechanism exaggerated by cisacting-elements (MCB elements) which ensure expression at the late G1-/S-phase of the cell cycle [1,16]. The RAD53 gene also contains two similar cis-acting-elements in its promoter that up-regulate its expression at late G1-/S-phase [17].…”

“…Two essential genes, MEC1 and RAD53, are central players in the kinase cascade that leads to cell-cycle arrest at all the checkpoints and transcriptional activation in response to DNA damage. Cells with mutations in the Mec1 or Rad53 genes are defective in both cell-cycle arrest and gene expression responses in response to DNA damage, but retain their essential functions for cell survival under normal conditions [1][2][3][4][5]. Among the best characterized transcriptional targets of the Rad53 kinase cascade are the RNR genes, which encode subunits of ribonucleotide reductase, the enzyme that catalyzes the rate-limiting step in dNTP synthesis [1].…”

“…Cells with mutations in the Mec1 or Rad53 genes are defective in both cell-cycle arrest and gene expression responses in response to DNA damage, but retain their essential functions for cell survival under normal conditions [1][2][3][4][5]. Among the best characterized transcriptional targets of the Rad53 kinase cascade are the RNR genes, which encode subunits of ribonucleotide reductase, the enzyme that catalyzes the rate-limiting step in dNTP synthesis [1]. In addition to transcriptional induction of RNR genes, the Rad53 pathway also induces RNR activity through the removal of Sml1, an RNR inhibitor that binds to large subunits and inhibits enzyme activity [6].…”

“…Although mutation of Sml1 has been shown to increase dNTP levels, it has never been established that mutation of Rad53 has a reciprocal effect on dNTP levels. Furthermore, Rad53 stimulates transcription of the genes required for dNTP synthesis, including RNR1, 2, and 4 [1]. Thus, mutation of Rad53 would also be expected to have a transcriptionally mediated inhibitory effect on dNTP levels.…”