title: S phase block following MEC1ATR inactivation occurs without severe dNTP depletion.
abstract: Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by chromosome breakage and cell death during G2/M. The S phase arrest is proposed to stem from a defect in Mec1-mediated degradation of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), causing a severe depletion in cellular dNTP pools. Here, the casual link between the S phase arrest, Sml1, and dNTP-levels is examined using a temperature sensitive mec1 mutant. In addition to S phase arrest, thermal inactivation of Mec1 leads to constitutively high levels of Sml1 and an S phase arrest. Expression of a novel suppressor, GIS2, a conserved mRNA binding zinc finger protein, rescues the arrest without down-regulating Sml1 levels. The dNTP pool in mec1 is reduced by ∼17% and GIS2 expression restores it, but only partially, to ∼93% of a control. We infer that the permanent S phase block following Mec1 inactivation can be uncoupled from its role in Sml1 down-regulation. Furthermore, unexpectedly modest effects of mec1 and GIS2 on dNTP levels suggest that the S phase arrest is unlikely to result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to small changes in its availability.
authors.affiliation: Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, MRC, London NW7 1AA, UK.
authors.forename: Caroline
authors.initials: C
authors.lastname: Earp
authors.affiliation: Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, MRC, London NW7 1AA, UK.
authors.forename: Samuel
authors.initials: S
authors.lastname: Rowbotham
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå SE 901 87, Sweden.
authors.forename: Gábor
authors.initials: G
authors.lastname: Merényi
authors.affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, Umeå SE 901 87, Sweden.
authors.forename: Andrei
authors.initials: A
authors.lastname: Chabes
authors.affiliation: Stem Cell Biology and Developmental Genetics, National Institute for Medical Research, MRC, London NW7 1AA, UK North West Cancer Research Institute, School of Medical Sciences, Bangor University, Bangor LL57 2UW, UK r.cha@bangor.ac.uk.
authors.forename: Rita S
authors.initials: RS
authors.lastname: Cha
keywords: ATR; DNA replication; Mec1; RNR; Replication arrest; Sml1; dNTP