A PP4-Phosphatase Complex Dephosphorylates γ-H2AX Generated during DNA Replication

Abstract

Summary The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce γ-H2AX. γ-H2AX stabilizes cell cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve γ-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2 and PP4R3β, that specifically dephosphorylates ATR-mediated γ-H2AX generated during DNA replication. PP4 efficiently dephosphorylates γ-H2AX within mononucleosomes in vitro. The effect of PP4 on γ-H2AX is independent of ATR and checkpoint kinase activity. When the PP4 complex is silenced, repair of DNA replication mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore γ-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation

“…The time course of H2AX phosphorylation in TPX2-depleted cells differs from those observed when specific ␥-H2AX protein phosphatases are depleted in which cases prolonged ␥-H2AX signals are observed (77)(78)(79)(80)(81)(82). In contrast, TPX2-depleted cells silence their elevated ␥-H2AX signals with the same time course as control cells (Fig.…”

Protein phosphatase PP4 is overexpressed in human breast and lung tumors

“…Since cisplatin is a replication-dependent DNA-damaging agent [8], this suggests that PP4 is involved in DNA damage signaling and/or repair. Indeed, we recently found that PP4C dephosphorylates γ-H2AX generated during DNA replication [9], while PP2A dephosphorylates γ-H2AX generated in response to exogenous DNA damaging agents [10].…”

“…Resistance may be attributed to several factors, including enhanced DNA repair, tolerance to platinum-induced DNA damage, and alterations in signal transduction pathways [8]. We recently demonstrated that PP4C is the phosphatase that dephosphorylates γ-H2AX generated during DNA replication [9]. High levels of PP4C in cells may dephosphorylate replication block-associated γ-H2AX, promote DNA replication regardless of replication block, escape cisplatin-induced apoptosis, and accumulate DNA mutations, which further predispose cells to genomic instability.…”

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

“…To date, the S. cerevisiae Ser/Thr phosphatases Ptc2 and Ptc3, as well as the protein phosphatase 4 (PP4)-like protein phosphatase Pph3, have been found to be important for checkpoint recovery after a single DSB (17,18). Furthermore, Pph3/PP4 is required for ␥H2A dephosphorylation, which contributes to recovery from a DSB-induced checkpoint (4,17,25). Finally, Pph3 and Ptc2 have been implicated in Rad53 dephosphorylation and hence deactivation during recovery from MMS exposure (26,32).…”