Persistent repair intermediates induce senescence.

Double-stranded DNA breaks activate a DNA damage checkpoint in G2 phase to trigger a cell cycle arrest, which can be reversed to allow for recovery. However, damaged G2 cells can also permanently exit the cell cycle, going into senescence or apoptosis, raising the question how an individual cell decides whether to recover or withdraw from the cell cycle. Here we find that the decision to withdraw from the cell cycle in G2 is critically dependent on the progression of DNA repair. We show that delayed processing of double strand breaks through HR-mediated repair results in high levels of resected DNA and enhanced ATR-dependent signalling, allowing p21 to rise to levels at which it drives cell cycle exit. These data imply that cells have the capacity to discriminate breaks that can be repaired from breaks that are difficult to repair at a time when repair is still ongoing.

MRE11 stability is regulated by CK2-dependent interaction with R2TP complex.

The MRN (MRE11-RAD50-NBS1) complex is essential for repair of DNA double-strand breaks and stalled replication forks. Mutations of the MRN complex subunit MRE11 cause the hereditary cancer-susceptibility disease ataxia-telangiectasia-like disorder (ATLD). Here we show that MRE11 directly interacts with PIH1D1, a subunit of heat-shock protein 90 cochaperone R2TP complex, which is required for the assembly of large protein complexes, such as RNA polymerase II, small nucleolar ribonucleoproteins and mammalian target of rapamycin complex 1. The MRE11-PIH1D1 interaction is dependent on casein kinase 2 (CK2) phosphorylation of two acidic sequences within the MRE11 C terminus containing serines 558/561 and 688/689. Conversely, the PIH1D1 phospho-binding domain PIH-N is required for association with MRE11 phosphorylated by CK2. Consistent with these findings, depletion of PIH1D1 resulted in MRE11 destabilization and affected DNA-damage repair processes dependent on MRE11. Additionally, mutations of serines 688/689, which abolish PIH1D1 binding, also resulted in decreased MRE11 stability. As depletion of R2TP frequently leads to instability of its substrates and as truncation mutation of MRE11 lacking serines 688/689 leads to decreased levels of the MRN complex both in ATLD patients and an ATLD mouse model, our results suggest that the MRN complex is a novel R2TP complex substrate and that their interaction is regulated by CK2 phosphorylation.

Checkpoint control and cancer.

DNA-damaging therapies represent the most frequently used non-surgical anticancer strategies in the treatment of human tumors. These therapies can kill tumor cells, but at the same time they can be particularly damaging and mutagenic to healthy tissues. The efficacy of DNA-damaging treatments can be improved if tumor cell death is selectively enhanced, and the recent application of poly-(ADP-ribose) polymerase inhibitors in BRCA1/2-deficient tumors is a successful example of this. DNA damage is known to trigger cell-cycle arrest through activation of DNA-damage checkpoints. This arrest can be reversed once the damage has been repaired, but irreparable damage can promote apoptosis or senescence. Alternatively, cells can reenter the cell cycle before repair has been completed, giving rise to mutations. In this review we discuss the mechanisms involved in the activation and inactivation of DNA-damage checkpoints, and how the transition from arrest and cell-cycle re-entry is controlled. In addition, we discuss recent attempts to target the checkpoint in anticancer strategies.

Nucleoporin NUP153 guards genome integrity by promoting nuclear import of 53BP1.

53BP1 is a mediator of DNA damage response (DDR) and a tumor suppressor whose accumulation on damaged chromatin promotes DNA repair and enhances DDR signaling. Using foci formation of 53BP1 as a readout in two human cell lines, we performed an siRNA-based functional high-content microscopy screen for modulators of cellular response to ionizing radiation (IR). Here, we provide the complete results of this screen as an information resource, and validate and functionally characterize one of the identified 'hits': a nuclear pore component NUP153 as a novel factor specifically required for 53BP1 nuclear import. Using a range of cell and molecular biology approaches including live-cell imaging, we show that knockdown of NUP153 prevents 53BP1, but not several other DDR factors, from entering the nuclei in the newly forming daughter cells. This translates into decreased IR-induced 53BP1 focus formation, delayed DNA repair and impaired cell survival after IR. In addition, NUP153 depletion exacerbates DNA damage caused by replication stress. Finally, we show that the C-terminal part of NUP153 is required for effective 53BP1 nuclear import, and that 53BP1 is imported to the nucleus through the NUP153-importin-ß interplay. Our data define the structure-function relationships within this emerging 53BP1-NUP153/importin-ß pathway and implicate this mechanism in the maintenance of genome integrity.

Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition.

DNA double-stranded breaks (DSBs) elicit a checkpoint response that causes a delay in cell cycle progression. Early in the checkpoint response, histone H2AX is phosphorylated in the chromatin region flanking the DSB by ATM/ATR and DNA-PK kinases. The resulting foci of phosphorylated H2AX (gamma-H2AX) serve as a platform for recruitment and retention of additional components of the checkpoint-signaling cascade that enhance checkpoint signaling and DSB repair. Upon repair, both the assembled protein complexes and the chromatin modifications are removed to quench the checkpoint signal. In this study, we show that the DNA damage-responsive Wip1 phosphatase is bound to chromatin. Moreover, Wip1 directly dephosphorylates gamma-H2AX and cells depleted of Wip1 fail to dephosphorylate gamma-H2AX during checkpoint recovery. Conversely, premature activation of Wip1 leads to displacement of MDC1 from damage foci and prevents activation of the checkpoint. Taken together, our data show that Wip1 has an essential role in dephosphorylation of gamma-H2AX to silence the checkpoint and restore chromatin structure once DNA damage is repaired.