Both DNA replication and DNA damage repair require
sufficient dNTP pools to facilitate new synthesis. In all organisms,
ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP
synthesis. In budding yeast, both in vivo and in vitro
experiments have shown that the activity of RNR is negatively regulated
by the binding of Sml1. Mutations in SML1 were first identified
as bypass suppressors of the essential functions of Mec1 and Rad53, two
kinases involved in DNA damage and replication checkpoints. To
understand the interplay amongst these genes, we measured the level of
Sml1 protein during the cell cycle and after DNA damage. Sml1 levels
fluctuate during the cell cycle-decreasing during S phase and peaking
during G2/M phase. This fluctuation is dependent on MEC1 and
RAD53. The temporary decrease of Sml1 levels during S phase is
crucial, since constitutive expression of Sml1 at this stage results in
cell death and blocks DNA synthesis in mec1 cells. After DNA
damage (MMS, UV or X-ray) as well as after HU treatment, Sml1 protein
also disappears. The diminution of Sml1 in response to these agents is
rapid and is completely abolished in mec1 and rad53 mutant
strains suggesting that the Mec1/Rad53 kinase cascade is required to
remove Sml1 protein after DNA damage. In support of this hypothesis, a
phosphorylated form of Sml1 transiently appears after DNA damage
concomitantly to its disappearance.
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