Ribonucleotide reductase (RNR) is a highly conserved enzyme that catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs, the rate-limiting step of deoxynucleoside triphosphate (dNTP) formation, and DNA synthesis. Its activity directly affects the balance and the levels of the dNTP pools and subsequently genetic stability (29
). Due to its vital importance, RNR is tightly regulated by both cell cycle and environmental cues. At S phase and after DNA damage, RNR activity is up-regulated to provide sufficient and balanced dNTP pools for DNA replication and repair. Mutations interfering with this regulated increase in RNR activity in yeast and humans can lead to growth defects and sensitivity to DNA-damaging agents (12
). RNR activity is also subjected to negative regulation, which is equally important. This is underscored by the observation that overexpression of a small subunit of yeast or human RNR in yeast cells causes chromosome instability (27
). Presumably, the deleterious effects of rampant RNR activity may be due to decreased DNA polymerase fidelity caused by excess dNTP levels and, at the same time, diminished NTP levels, which may interfere with RNA synthesis and numerous ATP/GTP-dependent cellular processes.
Currently, two mechanisms for RNR regulation are known. First, RNR is under allosteric control. In most organisms, the RNR holoenzyme is a tetramer composed of two distinct subunits (α2
), both of which contribute to the enzymatic activity. However, only the large subunit contains two allosteric sites: one regulates the balance among the four dNTP pools, and the other regulates feedback inhibition by monitoring the dATP/ATP ratio and modulating overall RNR activity accordingly (20
). Second, RNR is subjected to transcriptional regulation. In the budding yeast, transcription of the RNR
genes is induced in S phase and after DNA damage (10
). The induction in S phase is mediated by the MBP1/SW16
); the induction in response to DNA damage is controlled by the MEC1/RAD53
cell cycle checkpoint pathway (9
). This latter pathway can activate a downstream kinase, Dun1, which in turn relieves transcriptional repression by Crt1 (18
). In humans, transcription of the RNR large subunit and one RNR small subunit is also induced at S phase (28
). Recently, a new human RNR small subunit (p53R2) was shown to be induced by p53 after DNA damage. Moreover, the p53-dependent induction of p53R2 is crucial for DNA repair and cell survival after DNA damage (32
It is noteworthy that the conservation of RNR from yeast to humans extends beyond the sequence level to include the mechanism of transcriptional regulation via conserved DNA damage checkpoint pathways. Yeast Mec1 is a homolog of ATM (ataxia telangiectasia mutated) and ATR (ataxia- and Rad-related) in humans (16
); yeast Rad53 is the homolog of CHK2 in humans, which is mutated in some Li-Fraumeni syndrome patients (2
). Both ATM-ATR and CHK2 function upstream of p53 in the DNA damage response (3
). From an evolutionary perspective, the conservation of this pathway and its components underscores the importance of dNTP regulation in cell survival.
Recently, a protein inhibitor of RNR has been discovered in yeast. A study of a suppressor of mec1
) showed that the SML1
gene negatively regulates dNTP levels (35
). Furthermore, it was demonstrated that the Sml1 protein binds to a large subunit of RNR (Rnr1) in vivo and in vitro and inhibits RNR activity efficiently (5
). These results suggest a new mode of RNR regulation: Mec1 and Rad53 are required to relieve the Sml1-Rnr1 interaction in S phase, allowing synthesis of sufficient amounts of dNTPs for DNA replication. According to this model, in the absence of Mec1 or Rad53, decreased activity of RNR due to constitutive inhibition by Sml1 may cause insufficient dNTP levels and subsequent cell death (35
The aforementioned model suggests a new mode of RNR regulation and provides a simple explanation for the essential function of Mec1 and Rad53. However, based on current data, other possibilities cannot be excluded. In particular, is suppression of mec1
lethality by sml1
mutations really due to loss of RNR inhibition or is there yet another unidentified mechanism(s)? The existing sml1
alleles do not help differentiate between these possibilities since both are null mutations: one is a deletion of the SML1
open reading frame (ORF) (sml1
Δ) and the other is a deletion of its promoter (sml1-1
). Either mutation may abolish other unknown functions of Sml1 to relieve mec1
lethality. Additionally, other genetic suppressors of mec1
lethality, namely overexpression of RNR1
or deletion of the transcriptional repressor CRT1
), are also not informative, as they likely increase the amount of Rnr1 which titrates Sml1 activity.
Here, we address the above issue by reasoning that if inhibition of RNR by Sml1 leads to inviability in mec1 and rad53 cells, then loss-of-function missense mutations of Sml1 should either fail to interact with Rnr1 or abolish its RNR inhibitory activity. Therefore, we performed a comprehensive screening that permits the identification of any sml1 missense mutation that rescues mec1Δ and rad53Δ lethality but does not affect protein levels. Next, we asked whether such mutated forms of Sml1 could bind to Rnr1 and inhibit RNR activity. Using such an approach, we obtained seven sml1 missense mutations. Interestingly, all of these mutations mapped to the last 33 amino acid residues and they all abolish the Sml1-Rnr1 interaction in a two-hybrid assay. Moreover, four mutations were tested in an in vitro RNR assay, and all four no longer inhibit the enzyme. These results demonstrate that the loss of Sml1-Rnr1 interaction is sufficient to suppress mec1 and rad53 lethality.
In addition, the C-terminal clustering of seven Sml1 mutations suggests that this region may contain important structures. Investigation by nuclear magnetic resonance (NMR) studies revealed that the 104-amino-acid-residue-long Sml1 polypeptide has a loosely folded tertiary structure with an N- and a C-terminal alpha helix oriented in an antiparallel fashion. All seven sml1 missense mutations reside in or are adjacent to the C-terminal alpha helix. Deletion analysis further confirmed that only the C-terminal half of Sml1 is required for inhibition of RNR activity. Taken together, these in vivo and in vitro data define the Rnr1 interaction domain of Sml1. Interestingly, all seven sml1 mutations also abolished the interaction with the other yeast RNR large subunit (Rnr3) as well as with the human RNR large subunit, suggesting a conserved binding mechanism for these interactions.