Dephosphorylation of γH2A by Glc7/Protein Phosphatase 1 Promotes Recovery from Inhibition of DNA Replication

Abstract

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 (␥H2A) and dephosphorylates ␥H2A in vitro. Finally, the replication recovery defects of HU-treated glc7 mutants are partially rescued by Rad53 inactivation or lack of ␥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 ␥H2A dephosphorylation is a critical Glc7 function in this process.Eukaryotic cells require specialized surveillance mechanisms called checkpoints to preserve genome integrity in the presence of genotoxic insults. An efficient checkpoint response is also important during S phase, where it inhibits late origin firing, prevents stalled replication fork breakdown, and promotes the restart of replication (6,22,23,33,34). Checkpoint activation requires protein phosphorylation cascades that in Saccharomyces cerevisiae are initiated by the two protein kinases Mec1 (ATR in humans), which functions in a complex with Ddc2 (27), and Tel1 (ATM in humans) (reviewed in reference 20).Mec1 and Tel1 phosphorylate the central effector kinases Rad53 and Chk1, which transfer the arrest signal to a myriad of downstream proteins (reviewed in reference 20). Rad53 and Chk1 activation is not governed by their simple interaction with Mec1 or Tel1 but rather requires a stepwise process. Once recruited to the double-strand break (DSB) ends, Mec1 phosphorylates Rad9, which promotes the recruitment of inactive Rad53 in a forkhead-associated domain (FHA)-dependent manner, thus allowing its activating phosphorylation by Mec1 (31), as well as Rad53 in trans autophosphorylation, by increasing the local concentration of Rad53 molecules (14). Active Rad53 kinase molecules are then released from the complex and can phosphorylate downstream targets to arrest mitotic cell cycle progression. Mec1 activation is supported by independent loading onto DNA of the Ddc1-Rad17-Mec3 complex by Rad24-RFC, which enhances Mec1 ability to transmit and amplify the DNA damage signals (24).Mec1 and Tel1 also phosphorylate histone H2A on serine 129 (␥H2A) in response to DNA DSBs (12, 28, 30) and inhibition...

Serine/threonine phosphatases in the DNA damage response and cancer

“…Finally, even the Ptc2, Ptc3 and Pph3 triple-negative strain still efficiently removes Rad53 phosphorylation induced by replication stress, suggesting involvement of other phosphatase(s) under this condition (Travesa et al, 2008). Glc7/PP1 appears to be a plausible candidate, owing to the fact that it is required for dephosphorylation of Rad53 and recovery from the replication checkpoint (Bazzi et al, 2010). Despite this evidence, a potential direct connection between Rad53 and Glc7 remains to be verified.…”

“…Furthermore, Glc7, the PP1 homolog in Saccharomyces cerevisiae, dephosphorylates H2A at Ser 129 and is required for recovery from checkpoints induced by replication stress and DSBs (Bazzi et al, 2010). It would be interesting to confirm PP1-dependent regulation of g-H2AX in higher organisms and study whether an evolutionally conserved targeting subunit is responsible for this regulation.…”

“…It is thus possible that the increased g-H2AX level in PP2A-deficient cells is partly due to reduced repair. However, inhibition of PP4 by siRNA or depletion of Glc1/PP1 had no significant effect on DNA repair and knockdown of Wip1 by shRNA promoted repair of DNA DSBs (Bazzi et al, 2010;Moon et al, 2010).…”

Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex

“…Glc7 has been implicated in many cellular pathways, including glycogen metabolism (Feng et al 1991), control of budding (Black et al 1995), premeiotic DNA synthesis (Ramaswamy et al 1998), mitotic control (Hisamoto et al 1994;Black et al 1995), and recovery from checkpoint arrest (Bazzi et al 2010). Compared with protein kinases, little is known about the involvement of protein phosphatases in the regulation of DNA replication.…”

Chemical “Diversity” of Chromatin Through Histone Variants and Histone Modifications

“…Compared to histone kinases, histone phosphatases and phospho-histone-binding domains are less well studied and understood. Nevertheless, in addition to the previously identified H3S10 dephosphorylation by PP1 [197] and PP2A [198], more recent studies have identified phosphatases for H4S47ph (PP1 and Wip), H3T3ph/S10ph (mitogen activated protein kinase phosphatase 2), and H3T3ph (Repoman/PP1) [199][200][201][202][203][204][205]. Similarly, while earlier studies have identified several phospho-histone binders, such as specific isoforms of 14-3-3 for H3S10ph/S28ph and MDC1 for H2A.XS129ph, more recent studies have identified survivin (H3T3ph), WDR5 (H3T11ph), and shughosin (H2AS120ph) as phospho-histone readers [173,[206][207][208].…”