The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5 to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICLspecific incision on the 3-side of an ICL. The ICL-specific 3-incision, along with the 5-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork. DNA interstrand cross-links (ICLs)2 are formed as a result of DNA damage to each strand of the duplex. These lesions are located one or two nucleotides apart on the opposite strands and covalently link them, blocking DNA replication and transcription (1-3). Therefore, ICLs are considered to be the most cytotoxic DNA lesion (2-4).Several models for mammalian ICL repair have been proposed (2, 5-7). The first critical step for repairing ICLs in any given model is to release the ICL from one of the strands by making two incisions that bracket an ICL (unhooking the ICL). The unhooking of ICLs permits strand separation. In Escherichia coli and yeast, nucleotide excision repair (NER) makes dual incisions bracketing the ICL on one of the cross-linked strands to unhook ICLs (2, 3). Interestingly, NER is dispensable in mammalian ICL repair (5,8). In contrast to NER-defective mutants in E. coli and yeast, the NER-defective mammalian cells (except for ERCC-1 and ERCC-4 cells) are only moderately sensitive to DNA cross-linking agents such as mitomycin C (MMC) (8 -11). Moreover, biochemical studies demonstrated that the dual incisions by mammalian NER do not "unhook" ICLs (12, 13). Therefore, mammalian cells evidently have a unique mechanism(s) to initiate ICL repair.Notably, double strand break (DSB) repair-defective mutant cells, including those deficient in Rad51 paralogs or BRCA2, are hypersensitive to DNA cross-linking agents such as MMC (10). It has also been reported that ICLs in the mammalian genome are removed during S-phase and the DNA replication-mediated formation of a DSB is the key intermediate in ICL repair in mammalian cells (1,8,14,15). These data indicate that mammalian ICL repair goes through three critical processes: the formation of DNA replication-mediated DSBs, unhooking of the ICLs (separation of the two linked strands), and repair of the DSBs (restoration of the collapsed replication fork). It is unknown whether the formation of DSBs precedes the unhooking reaction. When the DNA replication complex encounters an ICL, the strand separat...

Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G 2 /M DNA damage checkpoint and efficient checkpoint recovery.Various types of DNA damage are frequently induced by both endogenous and exogenous agents, posing enormous threats to the cell and its genomic integrity. The cell responds to the occurrence of DNA damage by engaging DNA repair machineries to restore normal DNA structure and by activating the checkpoint mechanism through complex networks of signal transduction to halt cell cycle progression (1). Eventually, if the cell successfully repairs its damaged DNA, checkpoints are to be turned off to allow resumption of cell cycle progression. This process, termed "checkpoint recovery," is contrasted by permanent checkpoint arrest or programmed cell death (senescence or apoptosis, respectively), both of which are believed to result from unrepaired DNA damage and sustained DNA damage signaling (2, 3).The turn-off mechanism of the DNA damage checkpoint during recovery is poorly understood. Existing studies suggest that protein dephosphorylation and proteolysis are effective ways to deactivate checkpoint signaling. The involvement of numerous serine/threonine phosphatases in checkpoint recovery is not surprising given the crucial role of protein phosphorylation and kinase cascades in checkpoint activation (4). The wild-type p53-induced phosphatase Wip1 (PP2C␦ or PP...

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) plays a key role in mediating non-homologous end joining (NHEJ), a major repair pathway for DNA double-strand breaks (DSBs). The activation, function and dynamics of DNA-PKcs is regulated largely by its reversible phosphorylation at numerous residues, many of which are targeted by DNA-PKcs itself. Interestingly, these DNA-PKcs phosphorylation sites function in a distinct, and sometimes opposing manner, suggesting that they are differentially regulated via complex actions of both kinases and phosphatases. In this study we identified several phosphatase subunits as potential DSB-associated proteins. In particular, protein phosphatase 1 (PP1) is recruited to a DSB-mimicking substrate in Xenopus egg extracts and sites of laser microirradiation in human cells. Depletion of PP1 impairs NHEJ in both Xenopus egg extracts and human cells. PP1 binds multiple motifs of DNA-PKcs, regulates DNA-PKcs phosphorylation, and is required for DNA-PKcs activation after DNA damage. Interestingly, phosphatase 1 nuclear targeting subunit (PNUTS), an inhibitory regulator of PP1, is also recruited to DNA damage sites to promote NHEJ. PNUTS associates with the DNA-PK complex and is required for DNA-PKcs phosphorylation at Ser-2056 and Thr-2609. Thus, PNUTS and PP1 together fine-tune the dynamic phosphorylation of DNA-PKcs after DNA damage to mediate NHEJ.

These authors equally contributed to this work.Keywords: B55a, checkpoint recovery, DNA damage, Plk1, PP2AIn addition to governing mitotic progression, Plk1 also suppresses the activation of the G2 DNA damage checkpoint and promotes checkpoint recovery. Previous studies have shown that checkpoint activation after DNA damage requires inhibition of Plk1, but the underlying mechanism of Plk1 regulation was unknown. In this study we show that the specific phosphatase activity toward Plk1 Thr-210 in interphase Xenopus egg extracts is predominantly PP2A-dependent, and this phosphatase activity is upregulated by DNA damage. Consistently, PP2A associates with Plk1 and the association increases after DNA damage. We further revealed that B55a, a targeting subunit of PP2A and putative tumor suppressor, mediates PP2A/Plk1 association and Plk1 dephosphorylation. B55a and PP2A association is greatly strengthened after DNA damage in an ATM/ATR and checkpoint kinase-dependent manner. Collectively, we report a phosphatase-dependent mechanism that responds to DNA damage and regulates Plk1 and checkpoint recovery.

Greatwall (Gwl) kinase plays an essential role in the regulation of mitotic entry and progression. Mitotic activation of Gwl requires both cyclin-dependent kinase 1 (CDK1)-dependent phosphorylation and its autophosphorylation at an evolutionarily conserved serine residue near the carboxyl terminus (Ser-883 in ). In this study we show that Gwl associates with protein phosphatase 1 (PP1), particularly PP1γ, which mediates the dephosphorylation of Gwl Ser-883. Consistent with the mitotic activation of Gwl, its association with PP1 is disrupted in mitotic cells and egg extracts. During mitotic exit, PP1-dependent dephosphorylation of Gwl Ser-883 occurs prior to dephosphorylation of other mitotic substrates; replacing endogenous Gwl with a phosphomimetic S883E mutant blocks mitotic exit. Moreover, we identified PP1 regulatory subunit 3B (PPP1R3B) as a targeting subunit that can direct PP1 activity toward Gwl. PPP1R3B bridges PP1 and Gwl association and promotes Gwl Ser-883 dephosphorylation. Consistent with the cell cycle-dependent association of Gwl and PP1, Gwl and PPP1R3B dissociate in M phase. Interestingly, up-regulation of PPP1R3B facilitates mitotic exit and blocks mitotic entry. Thus, our study suggests PPP1R3B as a new cell cycle regulator that functions by governing Gwl dephosphorylation.