Dephosphorylation of gamma H2A by Glc7/protein phosphatase 1 promotes recovery from inhibition of DNA replication.
Replication fork stalling caused by deoxynucleotide depletion triggers Rad53 phosphorylation and subsequent checkpoint activation, which in turn play a crucial role in maintaining functional DNA replication forks. How cells regulate checkpoint deactivation after inhibition of DNA replication is poorly understood. Here, we show that the budding yeast protein phosphatase Glc7/protein phosphatase 1 (PP1) promotes disappearance of phosphorylated Rad53 and recovery from replication fork stalling caused by the deoxynucleoside triphosphate (dNTP) synthesis inhibitor hydroxyurea (HU). Glc7 is also required for recovery from a double-strand break-induced checkpoint, while it is dispensable for checkpoint inactivation during methylmethane sulfonate exposure, which instead requires the protein phosphatases Pph3, Ptc2, and Ptc3. Furthermore, Glc7 counteracts in vivo histone H2A phosphorylation on serine 129 (gamma H2A) and dephosphorylates gamma H2A in vitro. Finally, the replication recovery defects of HU-treated glc7 mutants are partially rescued by Rad53 inactivation or lack of gamma H2A formation, and the latter also counteracts hyperphosphorylated Rad53 accumulation. We therefore propose that Glc7 activity promotes recovery from replication fork stalling caused by dNTP depletion and that gamma H2A dephosphorylation is a critical Glc7 function in this process.
Bazzi
Marco
M
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milan, Italy.
Mantiero
Davide
D
Trovesi
Camilla
C
Lucchini
Giovanna
G
Longhese
Maria Pia
MP
D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006918:Hydroxyurea / Q000494:pharmacology; D009154:Mutation; D010766:Phosphorylation; D054645:Protein Phosphatase 1 / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D006657:Histones; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; C092048:GLC7 protein, S cerevisiae; D054645:Protein Phosphatase 1; D006918:Hydroxyurea