The basic machinery that detects DNA damage is the same throughout the cell cycle. Here, we show, in contrast, that reversal of DNA damage responses (DDRs) and recovery are fundamentally different in G1 and G2 phases of the cell cycle. We find that distinct phosphatases are required to counteract the checkpoint response in G1 vs. G2. Whereas WT p53-induced phosphatase 1 (Wip1) promotes recovery in G2-arrested cells by antagonizing p53, it is dispensable for recovery from a G1 arrest. Instead, we identify phosphoprotein phosphatase 4 catalytic subunit (PP4) to be specifically required for cell cycle restart after DNA damage in G1. PP4 dephosphorylates Krüppel-associated box domain-associated protein 1-S473 to repress p53-dependent transcriptional activation of p21 when the DDR is silenced. Taken together, our results show that PP4 and Wip1 are differentially required to counteract the p53-dependent cell cycle arrest in G1 and G2, by antagonizing early or late p53-mediated responses, respectively. A cell's genomic integrity is constantly challenged by endogenous and exogenous sources of DNA damage. Doublestrand breaks (DSBs) are particularly threatening to the genomic stability of proliferative cells and provoke a checkpoint response that coordinates repair processes with further cell cycle progression to prevent the replication and segregation of broken DNA. This DNA damage response (DDR) is orchestrated by multiple kinases that sense the DNA damage and relay this signal (1). Cellular recovery from a DNA damage insult ultimately requires the termination of the DDR once repair of the DNA is complete.PI3-kinase-related kinases (PIKKs), ataxia telangiectasia mutated (ATM), and ATM-and Rad3-related (ATR) are activated by distinct structures of damaged DNA and phosphorylate histone H2AX in the vicinity of the damaged site to recruit repair proteins (2). In addition to such local events, ATM and ATR activate a subsequent layer of checkpoint kinase 2 (Chk2) and Chk1, respectively, that disseminates from the damaged site (3, 4). ATM also activates p38 mitogen-activated protein kinase (MAPK), which coordinates the DDR outside the nucleus (5, 6). Combined, these checkpoint kinases ensure that cell cycle progression is prevented at the G1/S or G2/M boundary (7).PIKKs and checkpoint kinases commonly converge on the transcription factor p53, a key regulator of stress responses. Phosphorylation of p53 prevents its degradation by mouse double minute 2 (Mdm2)-mediated polyubiquitination, allowing p53 to accumulate and induce its target genes, including p21 (1). Both p53 and its transcriptional target p21 are sufficient to impose an arrest in both G1 and G2, and they are absolutely required for a bona fide checkpoint arrest in G1 (8-11).Recovery from a checkpoint-induced arrest requires silencing of the checkpoint machinery and coincides with the removal of phosphorylations deposited by PIKKs and other checkpoint kinases. We have previously shown that WT p53-induced phosphatase 1 (Wip1) is essential for checkpoint recovery from a...

Activation of the DNA-damage checkpoint culminates in the inhibition of cyclin-dependent kinase (Cdk) complexes to prevent cell-cycle progression. We have shown recently that Cdk activity is required for activation of the Forkhead transcription factor FoxM1, an important regulator of gene expression in the G2 phase of the cell cycle. Here, we show that FoxM1 is transcriptionally active during a DNA-damage-induced G2 arrest and is essential for checkpoint recovery. Paradoxically, Cdk activity, although reduced after checkpoint activation, is required to maintain FoxM1-dependent transcription during the arrest and for expression of pro-mitotic targets such as cyclin A, cyclin B and Plk1. Indeed, we find that cells need to retain sufficient levels of Cdk activity during the DNA-damage response to maintain cellular competence to recover from a DNA-damaging insult.

Polo-like kinase 1 (Plk1) is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2, and at the end of mitosis, it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here, we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function.