LAUSR

The DNA damage checkpoint is a signal transduction cascade with three layers of kinases. Checkpoint kinases are conventionally defined as kinases that are activated by DNA damage— either directly or by upstream kinases—and phosphorylate targets that preserve genome stability. In the presence of damaged DNA, the sensor kinases (ATM/ATR in mammals and Tel1/ Mec1 in budding yeast) become active and phosphorylate the effector kinases (CHK1 and CHK2 in mammals and Chk1 and Rad53 in budding yeast [1]). Rad53, in turn, activates another checkpoint kinase, Dun1 [2]. This hierarchy is likely to provide both amplification and specialization, as the substrates of each set of kinases are selectively enriched for proteins in particular areas of biology. ATM and ATR are localized to DNA breaks, and their substrates are enriched for chromatin components and repair proteins that are similarly localized (e.g., H2AX and Slx4) [3–7]. While Rad53, Chk1, and their homologues target some proteins in this category, they primarily act on a large number of substrates that are not directly adjacent to sites of DNA damage, including cell cycle regulators, such as Sld3 [8, 9] and Pds1 [10]. By contrast, Dun1’s only known substrates are involved in the regulation of ribonucleotide levels [11]. Here, Liu and colleagues [12] show that the GSK3-related kinase Mck1 is directly activated by Rad53 and, like Dun1, regulates ribonucleotide biosynthesis, suggesting it too is a checkpoint kinase. All eukaryotic organisms require an adequate concentration of deoxyribonucleoside triphosphates (dNTPs) in order to ensure accurate DNA replication and repair and to maintain genomic stability. The rate-limiting step in dNTP synthesis is catalyzed by ribonucleotide reductase (RNR), an essential heterotetrameric enzyme that mediates the reduction of ribonucleotides (rNTPs) into deoxyribonucleotides (dNTPs). In the budding yeast Saccharomyces cerevisiae, the large R1 subunit is composed of an Rnr1 homodimer (or Rnr1-Rnr3 heterodimer), whereas the active small R2 subunit is formed by an Rnr2-Rnr4 heterodimer [13]. The activity of RNR is tightly regulated by the cell cycle and environmental cues, which is critical since an unbalanced supply of dNTPs dramatically increases the mutation rate. Once Dun1 becomes activated, it enhances RNR activity by multiple mechanisms. First, in response to DNA damage and replication stress, Dun1 phosphorylates the Crt1 repressor. This causes it to be lost from RNR promoters, leading to an increase in transcription of RNR2, RNR3, and RNR4. However, induction of RNR genes upon genotoxic stress is not completely dependent upon the Dun1 kinase. In dun1Δmutants, RNR genes continue to be significantly induced in response to DNA damage [14]. DUN1-independent RNR1 induction upon DNA damage is also Crt1-independent. This is mediated by Rad53 activation of Ixr1, a DNA-binding protein that interacts with the RNR1 promoter and activates RNR1 transcription [15]. In addition, the yeast Rnr1 inhibitor Sml1 undergoes a DUN1-dependent phosphorylation that leads to Sml1