TY - JOUR

T1 - Acute sensitivity of DNA replication to reduction in dNTP pools following Mec1ATR inactivation

AU - Earp, C.

AU - Rowbotham, S.

AU - Marjavaara, L.

AU - Chabes, A.

AU - Cha, R.S.

PY - 2015/10/16

Y1 - 2015/10/16

N2 - Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by a fatal mitotic catastrophe. The mec1 S phase arrest was proposed to stem from a defect in the Mec1-Rad53-Dun1 dependent removal of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), at the onset of S phase: According to this view, Sml1 removal and the ensuing RNR activation would promote the dNTP production necessary for genome duplication. In support for this view, dNTP levels in hypomorphic mec1 or rad53 mutants and a dun1∆ strain were shown to be reduced by as much as 46% compared to a MEC1 control strain. Notably however, nearly all analyses on a lethal mec1 allele (e.g. mec1∆ or mec1-kd [kinase dead]) have been performed in a strain background that was either deleted for SML1 or over-expressing RNR1, a requirement for maintaining viability of a mutant lacking Mec1's essential function. As a result, while it is clear that absence of Mec1 causes dNTP pool to decrease, the true extent of the reduction and whether it would be sufficient to account for the replication arrest remain elusive. Here, we addressed these questions utilizing a temperature sensitive mutant, mec1-4, which maintains its viability at permissive temperature in an otherwise wild-type background, circumventing the need to exogenously manipulate Sml1 and/or RNR activity. Results show that Mec1 inactivation leads to an S phase arrest and a ~17% reduction in dNTP pool; expression of a novel suppressor, GIS2 (glucose inhibition of gluconeogenic growth suppressor 2), rescues the arrest and partially restores the dNTP pool to ~ 93% of a control. Unexpectedly modest effects of mec1 and GIS2 on dNTP levels demonstrate that the arrest does result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to its availability

AB - Inactivation of Mec1, the budding yeast ATR, results in a permanent S phase arrest followed by a fatal mitotic catastrophe. The mec1 S phase arrest was proposed to stem from a defect in the Mec1-Rad53-Dun1 dependent removal of Sml1, a conserved inhibitor of ribonucleotide reductase (RNR), at the onset of S phase: According to this view, Sml1 removal and the ensuing RNR activation would promote the dNTP production necessary for genome duplication. In support for this view, dNTP levels in hypomorphic mec1 or rad53 mutants and a dun1∆ strain were shown to be reduced by as much as 46% compared to a MEC1 control strain. Notably however, nearly all analyses on a lethal mec1 allele (e.g. mec1∆ or mec1-kd [kinase dead]) have been performed in a strain background that was either deleted for SML1 or over-expressing RNR1, a requirement for maintaining viability of a mutant lacking Mec1's essential function. As a result, while it is clear that absence of Mec1 causes dNTP pool to decrease, the true extent of the reduction and whether it would be sufficient to account for the replication arrest remain elusive. Here, we addressed these questions utilizing a temperature sensitive mutant, mec1-4, which maintains its viability at permissive temperature in an otherwise wild-type background, circumventing the need to exogenously manipulate Sml1 and/or RNR activity. Results show that Mec1 inactivation leads to an S phase arrest and a ~17% reduction in dNTP pool; expression of a novel suppressor, GIS2 (glucose inhibition of gluconeogenic growth suppressor 2), rescues the arrest and partially restores the dNTP pool to ~ 93% of a control. Unexpectedly modest effects of mec1 and GIS2 on dNTP levels demonstrate that the arrest does result from a severe depletion of dNTP pool as assumed, but a heightened sensitivity to its availability

UR - http://urn.kb.se/r0749-503X

M3 - Article

VL - 32

SP - S56

JO - Yeast

JF - Yeast

SN - 0749-503X

ER -