Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G₁/S checkpoint, ATM and p53 signaling pathways in p53-wild-type cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G₁/S checkpoint pathway in p53 wild-type lines and not in p53-mutant cells. These responses are coupled with G₂/G₁ checkpoint effectors p21^CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G₂ cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wild-type and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G₂ arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wild-type and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.Key words: DNA damaging agent, G₂ arrest, microarray, PARP inhibition, p53, topotecan, veliparib (ABT-888)     

HP1 promotes tumor suppressor BRCA1 functions during the DNA damage response

The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G₂/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.     

Nek1 kinase functions in DNA damage response and checkpoint control through a pathway independent of ATM and ATR

Never-in-mitosis A related protein kinase 1 (Nek1) is involved early in a DNA damage sensing/repair pathway. We have previously shown that cells without functional Nek1 fail to activate the more distal kinases Chk1 and Chk2 and fail to arrest properly at G₁/S or M-phase checkpoints in response to DNA damage. As a consequence, foci of damaged DNA in Nek1 null cells persist long after the instigating insult, and Nek1 null cells develop unstable chromosomes at a rate much higher than identically cultured wild-type cells. Here we show that Nek1 functions independently of canonical DNA damage responses requiring the PI3 kinase-like proteins ATM and ATR. Chemical inhibitors of ATM/ATR or mutation of the genes that encode them fail to alter the kinase activity of Nek1 or its localization to nuclear foci of DNA damage. Moreover ATM and ATR activities, including the localization of the proteins to DNA damage sites and phosphorylation of early DNA damage response substrates, are intact in Nek1^−/− murine cells and in human cells with Nek1 expression silenced by siRNA. Our results demonstrate that Nek1 is important for proper checkpoint control and characterize for the first time a DNA damage response that does not directly involve one of the known upstream mediator kinases, ATM or ATR.Key words: checkpoint control, DNA damage response, Nek1, ATM, ATR     

The Saccharomyces cerevisiae RAD9 cell cycle checkpoint gene is required for optimal repair of UV-induced pyrimidine dimers in both G(1) and G(2)/M phases of the cell cycle

Cells respond to DNA damage by activating both cellular growth arrest and DNA repair processes. In Saccharomyces cerevesiae the RAD9 gene controls DNA damage-mediated cell cycle arrest that is known to allow efficient repair. To ascertain whether RAD9 plays a role in DNA repair per se, the removal of UV-induced photolesions was assessed in synchronized isogenic normal and rad9Δ cells using the high resolution primer extension technique. The results show that RAD9 is indeed involved in the removal of photolesions from both the transcribed and the non-transcribed strands of the reporter GAL10 gene, in G₁- as well as G₂/M-arrested cells. Interestingly, these data also reveal that in both normal and rad9 mutant, the repair strand bias towards the transcribed stand is more pronounced in G₂/M- than in G₁-arrested cells. These data indicate that RAD9 coordinate the cellular response to DNA damage by activating both cell cycle checkpoint and excision repair.     

p16INK4A Participates in a G1 Arrest Checkpoint in Response to DNA Damage

Members of the INK4 protein family specifically inhibit cyclin-dependent kinase 4 (cdk4) and cdk6-mediated phosphorylation of the retinoblastoma susceptibility gene product (Rb). p16^INK4A, a prototypic INK4 protein, has been identified as a tumor suppressor in many human cancers. Inactivation of p16^INK4A in tumors expressing wild-type Rb is thought to be required in order for many malignant cell types to enter S phase efficiently or to escape senescence. Here, we demonstrate another mechanism of tumor suppression by implicating p16^INK4A in a G₁ arrest checkpoint in response to DNA damage. Calu-1 non-small cell lung cancer cells, which retain Rb and lack p53, do not arrest in G₁ following DNA damage. However, engineered expression of p16^INK4A at levels compatible with cell proliferation restores a G₁ arrest checkpoint in response to treatment with γ-irradiation, topoisomerase I and II inhibitors, and cisplatin. A similar checkpoint can be demonstrated in p53^−/− fibroblasts that express p16^INK4A. DNA damage-induced G₁ arrest, which requires the expression of pocket proteins such as Rb, can be abrogated by overexpression of cdk4, kinase-inactive cdk4 variants capable of sequestering p16^INK4A, or a cdk4 variant incapable of binding p16^INK4A. After exposure to DNA-damaging agents, there was no change either in overall levels of p16^INK4A or in amounts of p16^INK4A found in complex with cdks 4 and 6. Nonetheless, p16^INK4A expression is required for the reduction in cdk4- and cdk6-mediated Rb kinase activity observed in response to DNA damage. During tumor progression, loss of p16^INK4A expression may be necessary for cells with wild-type Rb to bypass this G₁ arrest checkpoint and attain a fully transformed phenotype.     

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1 ^−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1 ^−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1 ^−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1 ^−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2''s function and as a result leads to infertility and genomic instability in mice.     

PARG dysfunction enhances DNA double strand break formation in S-phase after alkylation DNA damage and augments different cell death pathways

Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg^−/− mouse ES cells and human cancer cell lines. Parg^−/− mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg^−/− mouse ES cells. Parg^−/− ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.     

53BP1, BRCA1, and the Choice between Recombination and End Joining at DNA Double-Strand Breaks

When DNA double-strand breaks occur, the cell cycle stage has a major influence on the choice of the repair pathway employed. Specifically, nonhomologous end joining is the predominant mechanism used in the G₁ phase of the cell cycle, while homologous recombination becomes fully activated in S phase. Studies over the past 2 decades have revealed that the aberrant joining of replication-associated breaks leads to catastrophic genome rearrangements, revealing an important role of DNA break repair pathway choice in the preservation of genome integrity. 53BP1, first identified as a DNA damage checkpoint protein, and BRCA1, a well-known breast cancer tumor suppressor, are at the center of this choice. Research on how these proteins function at the DNA break site has advanced rapidly in the recent past. Here, we review what is known regarding how the repair pathway choice is made, including the mechanisms that govern the recruitment of each critical factor, and how the cell transitions from end joining in G₁ to homologous recombination in S/G₂.     

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint

Dovitinib (TKI258; formerly CHIR‐258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G₂/M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single‐cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G₂ arrest similar to the G₂ DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ‐H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G₂ arrest could be overcome by abrogation of the G₂ DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib‐mediated G₂ cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP‐ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.     

Cutting edge: Chk1 directs senescence and mitotic catastrophe in recovery from G2 checkpoint arrest

Besides the well‐understood DNA damage response via establishment of G₂ checkpoint arrest, novel studies focus on the recovery from arrest by checkpoint override to monitor cell cycle re‐entry. The aim of this study was to investigate the role of Chk1 in the recovery from G₂ checkpoint arrest in HCT116 (human colorectal cancer) wt, p53^–/– and p21^–/– cell lines following H₂O₂ treatment. Firstly, DNA damage caused G₂ checkpoint activation via Chk1. Secondly, overriding G₂ checkpoint led to (i) mitotic slippage, cell cycle re‐entry in G₁ and subsequent G₁ arrest associated with senescence or (ii) premature mitotic entry in the absence of p53/p21^WAF1 causing mitotic catastrophe. We revealed subtle differences in the initial Chk1‐involved G₂ arrest with respect to p53/p21^WAF1: absence of either protein led to late G₂ arrest instead of the classic G₂ arrest during checkpoint initiation, and this impacted the release back into the cell cycle. Thus, G₂ arrest correlated with downstream senescence, but late G₂ arrest led to mitotic catastrophe, although both cell cycle re‐entries were linked to upstream Chk1 signalling. Chk1 knockdown deciphered that Chk1 defines long‐term DNA damage responses causing cell cycle re‐entry. We propose that recovery from oxidative DNA damage‐induced G₂ arrest requires Chk1. It works as cutting edge and navigates cells to senescence or mitotic catastrophe. The decision, however, seems to depend on p53/p21^WAF1. The general relevance of Chk1 as an important determinant of recovery from G₂ checkpoint arrest was verified in HT29 colorectal cancer cells.     

NAR Breakthrough Article: Helq acts in parallel to Fancc to suppress replication-associated genome instability

HELQ is a superfamily 2 DNA helicase found in archaea and metazoans. It has been implicated in processing stalled replication forks and in repairing DNA double-strand breaks and inter-strand crosslinks. Though previous studies have suggested the possibility that HELQ is involved in the Fanconi anemia (FA) pathway, a dominant mechanism for inter-strand crosslink repair in vertebrates, this connection remains elusive. Here, we investigated this question in mice using the Helq^gt and Fancc⁻ strains. Compared with Fancc^−/− mice lacking FANCC, a component of the FA core complex, Helq^gt/gt mice exhibited a mild of form of FA-like phenotypes including hypogonadism and cellular sensitivity to the crosslinker mitomycin C. However, unlike Fancc^−/− primary fibroblasts, Helq^gt/gt cells had intact FANCD2 mono-ubiquitination and focus formation. Notably, for all traits examined, Helq was non-epistatic with Fancc, as Helq^gt/gt;Fancc^−/− double mutants displayed significantly worsened phenotypes than either single mutant. Importantly, this was most noticeable for the suppression of spontaneous chromosome instability such as micronuclei and 53BP1 nuclear bodies, known consequences of persistently stalled replication forks. These findings suggest that mammalian HELQ contributes to genome stability in unchallenged conditions through a mechanism distinct from the function of FANCC.     

Differential effects of caffeine on DNA damage and replication cell cycle checkpoints in the fission yeast Schizosaccharomyces pombe

Caffeine potentiates the lethal effects of ultraviolet and ionising radiation on wild-type Schizosaccharomyces pombe cells. In previous studies this was attributed to the inhibition by caffeine of a novel DNA repair pathway in S. pombe that was absent in the budding yeast Saccharomyces cerevisiae. Studies with radiation-sensitive S. pombe mutants suggested that this caffeine-sensitive pathway could repair ultraviolet radiation damage in the absence of nucleotide excision repair. The alternative pathway was thought to be recombinational and to operate in the G₂ phase of the cell cycle. However, in this study we show that cells held in G₁ of the cell cycle can remove ultraviolet-induced lesions in the absence of nucleotide excision repair. We also show that recombination-defective mutants, and those now known to define the alternative repair pathway, still exhibit the caffeine effect. Our observations suggest that the basis of the caffeine effect is not due to direct inhibition of recombinational repair. The mutants originally thought to be involved in a caffeine-sensitive recombinational repair process are now known to be defective in arresting the cell cycle in S and/or G₂ following DNA damage or incomplete replication. The gene products may also have an additional role in a DNA repair or damage tolerance pathway. The effect of caffeine could, therefore, be due to interference with DNA damage checkpoints, or inhibition of the DNA damage repair/tolerance pathway. Using a combination of flow cytometric analysis, mitotic index analysis and fluorescence microscopy we show that caffeine interferes with intra-S phase and G₂ DNA damage checkpoints, overcoming cell cycle delays associated with damaged DNA. In contrast, caffeine has no effect on the DNA replication S phase checkpoint in reponse to inhibition of DNA synthesis by hydroxyurea.     

The USP1/UAF1 complex promotes double-strand break repair through homologous recombination

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1^−/−, UAF1^−/−/−, and USP1^−/− UAF1^−/−/− chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.     

Role of ATM and the Damage Response Mediator Proteins 53BP1 and MDC1 in the Maintenance of G2/M Checkpoint Arrest

ATM-dependent initiation of the radiation-induced G₂/M checkpoint arrest is well established. Recent results have shown that the majority of DNA double-strand breaks (DSBs) in G₂ phase are repaired by DNA nonhomologous end joining (NHEJ), while ∼15% of DSBs are slowly repaired by homologous recombination. Here, we evaluate how the G₂/M checkpoint is maintained in irradiated G₂ cells, in light of our current understanding of G₂ phase DSB repair. We show that ATM-dependent resection at a subset of DSBs leads to ATR-dependent Chk1 activation. ATR-Seckel syndrome cells, which fail to efficiently activate Chk1, and small interfering RNA (siRNA) Chk1-treated cells show premature mitotic entry. Thus, Chk1 significantly contributes to maintaining checkpoint arrest. Second, sustained ATM signaling to Chk2 contributes, particularly when NHEJ is impaired by XLF deficiency. We also show that cells lacking the mediator proteins 53BP1 and MDC1 initially arrest following radiation doses greater than 3 Gy but are subsequently released prematurely. Thus, 53BP1^−/− and MDC1^−/− cells manifest a checkpoint defect at high doses. This failure to maintain arrest is due to diminished Chk1 activation and a decreased ability to sustain ATM-Chk2 signaling. The combined repair and checkpoint defects conferred by 53BP1 and MDC1 deficiency act synergistically to enhance chromosome breakage.DNA double-strand breaks (DSBs) activate the DNA damage response (DDR), a coordinated process that functions to enhance survival and maintain genomic stability. The DDR includes pathways of DSB repair and a signal transduction response that activates apoptosis and cell cycle checkpoint arrest and influences DSB repair (15). DNA nonhomologous end joining (NHEJ) and homologous recombination (HR) represent the major DSB repair mechanisms, NHEJ being the major mechanism in G₀/G₁, while both processes function in G₂ (9, 32). Ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) are related phosphoinositol 3-kinase-like kinases (PIKKs) that regulate the DNA damage signaling response. ATM is activated by DSBs, while ATR is activated at single-strand (ss) regions of DNA via a process that involves ATRIP-replication protein A (RPA)-ssDNA association. Ionizing radiation (IR) induces DSBs, base damage, and ss nicks. Since neither base damage nor ss nicks activate ATR, IR-induced signaling in the G₁ and G₂ phases is predominantly ATM dependent (3, 29). In S phase, ATR can be activated by both endogenous and exogenously induced lesions following replication fork stalling/collapse (8).Recent work has shown that in G₂ phase, DSBs can undergo resection via an ATM-dependent process generating ssDNA regions that can activate ATR following RPA association (11). ATR activation at resected DSBs is coupled to loss of ATM activation (11). Although ATM and ATR share overlapping substrates, there is specificity in their signaling to the transducer kinases; ATM uniquely phosphorylates Chk2, while ATR phosphorylates Chk1. Phosphorylation of either Chk1 or Chk2 causes their activation. Critical targets of Chk1/Chk2 are the Cdc25 phosphatases, which regulate the cyclin-dependent kinases (Cdks), including Cdk1, the regulator of mitotic entry (18). Collectively, these studies suggest that two components of ATM-dependent signaling to the G₂/M checkpoint machinery can occur: ATM-Chk2 signaling at unresected DSBs and ATM-ATR-Chk1 signaling at resected DSBs.Although much is known about the mechanism leading to G₂/M checkpoint activation, few studies have addressed how arrest is maintained and how release coordinates with the status of DSB repair. We examine here the maintenance of checkpoint arrest during the immediate phase of DSB repair. We do not address the issue of checkpoint adaptation, a distinct phenomenon which occurs after prolonged checkpoint arrest (22). Further, we focus on the process maintaining arrest in irradiated G₂-phase cells and do not consider how arrest is maintained in irradiated S-phase cells that progress into G₂ phase. (Previous studies have shown that while G₂/M arrest is ATM dependent at early times post-IR, at later times it becomes ATR dependent as S-phase cells progress into G₂ phase [2, 33].) To focus on mechanisms maintaining ATM-dependent signaling in G₂-phase cells, we use aphidicolin (APH) to prevent S-phase cells from progressing into G₂ during analysis. We, thus, examine checkpoint maintenance in cells irradiated in G₂ phase and do not evaluate arrest regulated by ATR following replication fork stalling. The basis for our work stems from two recent advances. First, we evaluate the impact of ATM-mediated ATR activation in the light of recent findings that resection occurs in G₂ phase (11). Second, we consider the finding that NHEJ represents the major DSB repair mechanism in G₂ and that a 15 to 20% subset of DSBs, representing those that are rejoined with slow kinetics in an ATM-dependent manner, undergo resection and repair by HR (3, 25). Thus, contrary to the notion that HR represents the major DSB repair pathway in G₂ phase, it repairs only 15 to 20% of X- or gamma-ray-induced DSBs and represents the slow component of DSB repair in G₂ phase. Given these findings, several potential models for how checkpoint arrest is maintained in G₂ can be envisaged. A simple model is that the initial signal generated by IR is maintained for a defined time to allow for DSB repair. Such a model appears to explain the kinetics of checkpoint signaling in fission yeast after moderate IR (17). In mammalian cells, the duration of arrest depends on dose and DSB repair capacity (6). Thus, it is possible that the status of ongoing repair is communicated to the checkpoint machinery to coordinate timely release with the process of DSB repair. Here, we consider the impact of resection leading to ATM-ATR-Chk1 signaling versus ATM-Chk2 signaling from nonresected DSBs and how they interplay to maintain rather than initiate checkpoint arrest.Mediator proteins, including 53BP1 and MDC1, assemble at DSBs in an ATM-dependent manner, but their roles in the DDR are unclear. Cells lacking 53BP1 or MDC1 are proficient in checkpoint initiation after moderate IR doses, leading to the suggestion that these proteins are required for amplification of the ATM signal after exposure to low doses but are dispensable after high doses, when a robust signal is generated, even in their absence (7, 16, 28, 31). Despite their apparent subtle role in ATM signaling, cells lacking these mediator proteins display significant genomic instability (19). We thus also examine whether the mediator proteins contribute to the maintenance of checkpoint arrest.We identify two ATM-dependent processes that contribute to the maintenance of checkpoint arrest in G₂-phase cells: (i) ATR-Chk1 activation at resected DSBs and (ii) a process that involves sustained signaling from ATM to Chk2 at unrepaired DSBs. Further, we show that 53BP1 and MDC1 are required for maintaining checkpoint arrest, even following exposure to high radiation doses due to roles in ATR-Chk1 activation and sustained ATM-Chk2 signaling, and that this contributes to their elevated genomic instability.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.     

Suppression of BRCA1 sensitizes cells to proteasome inhibitors

BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity.BRCA1 is an important tumor suppressor gene whose germ-line or somatic inactivation is implicated in a significant number of breast and ovarian cancers.¹ Human BRCA1 encodes an 1863 amino-acid-long protein with a RING-finger domain at the N terminus and two BRCT domains located at the C terminus.^{2, 3} BRCT domains mediate interaction with phosphorylated proteins such as Abraxas, BACH1, CtIP and others involved in sensing DNA damage and assembly of the BRCA1-associated genome surveillance complex at sites of DNA breaks.⁴ The RING domain constitutively interacts with the BRCA1-associated RING domain protein (BARD1), forming a heterodimer having an E3 ubiquitin ligase activity.⁵ Ubiquitination of target proteins, including cell cycle or DNA repair-regulating proteins (e.g. CtIP (RBBP8), nucleophosmin (NPM1, B23), claspin (CLSPN) and others), occurs either at Lys48 residue of the ubiquitin leading to the 26S proteasome-mediated degradation of target proteins or at Lys6 or Lys63 having a trafficking and signaling role.⁶ A serine cluster coiled-coil domain spanning amino acids 1280–1524 contains multiple phosphorylation sites for ATM and ATR kinases activated by DNA damage.⁷ The same region also binds PALB2 protein linking BRCA1 to another major breast cancer predisposition gene BRCA2.⁸The most prominent function of BRCA1 is associated with its role in repair of DNA damage, particularly of double-stranded DNA breaks (DSBs), one of the most severe types of DNA lesions.⁹ BRCA1 is recruited to sites of DNA damage via a series of phosphorylation and ubiquitination events, where it serves as a binding scaffold for other DNA repair proteins,^{10, 11} ubiquitinates claspin, cyclin B and CDC25C, triggering cell cycle arrest to allow time for repair,¹² and facilitates BRCA2-mediated loading of RAD51 recombinase to enable the homologous recombination (HR) mechanism of DNA repair.⁹ In addition, BRCA1 may contribute to maintaining genome integrity by stabilizing the heterochromatin structure via ubiquitination of histone H2A.¹³ BRCA1 is also required for centrosome-dependent and -independent mitotic spindle formation, providing another route, by which loss of BRCA1 could promote chromosome instability and tumor formation.^{14, 15}Such a critical role of BRCA1 in DNA repair is exploited therapeutically. DNA-damaging agents, particularly DNA-crosslinking agents such as platinum-containing drugs, or ionizing radiation lead to the accumulation of DNA breaks requiring HR for repair and, therefore, are particularly toxic to BRCA1-deficient tumor cells.¹⁶ Pharmacological inhibitors of poly-(ADP-ribose) polymerases (PARPs) selectively kill BRCA1-deficient cells owing to defective HR, functioning as a back-up repair mechanism in the absence of the PARP-mediated repair of single-stranded DNA breaks.¹⁷ However, multiple mechanisms allow BRCA1-deficient cells to develop resistance to these drugs including elevated expression of the efflux transporters pumping the drugs out of the cell, secondary mutations restoring a functional BRCA1 protein and loss of 53BP1 protein, which counteracts BRCA1 and HR by blocking resection of DNA ends around the breaks (see Lord and Ashworth¹⁸ for the latest review). Therefore, additional efforts to identify small-molecule agents especially targeting BRCA1 functions unrelated to its DNA repair function are warranted.Here we performed a high-throughput chemical screen of BRCA1-depleted MDA-MB-231 cells using a collection of 198 US Food and Drug Administration (FDA)-approved and experimental drugs. We found that 26S proteasome inhibitors were more toxic to BRCA1 knockdown than control cells. Response of BRCA1-deficient cells to bortezomib involved deregulation of the RB1-mediated cell cycle checkpoint, activation of a noncanonical ERN1-mediated unfolded protein response and 53BP1-related G2/M cell cycle arrest. Our results reveal novel aspects of BRCA1 function unrelated to DNA repair.     

APC/CCdh1 controls CtIP stability during the cell cycle and in response to DNA damage

Human cells have evolved elaborate mechanisms for responding to DNA damage to maintain genome stability and prevent carcinogenesis. For instance, the cell cycle can be arrested at different stages to allow time for DNA repair. The APC/C^Cdh1 ubiquitin ligase mainly regulates mitotic exit but is also implicated in the DNA damage‐induced G₂ arrest. However, it is currently unknown whether APC/C^Cdh1 also contributes to DNA repair. Here, we show that Cdh1 depletion causes increased levels of genomic instability and enhanced sensitivity to DNA‐damaging agents. Using an integrated proteomics and bioinformatics approach, we identify CtIP, a DNA‐end resection factor, as a novel APC/C^Cdh1 target. CtIP interacts with Cdh1 through a conserved KEN box, mutation of which impedes ubiquitylation and downregulation of CtIP both during G₁ and after DNA damage in G₂. Finally, we find that abrogating the CtIP–Cdh1 interaction results in delayed CtIP clearance from DNA damage foci, increased DNA‐end resection, and reduced homologous recombination efficiency. Combined, our results highlight the impact of APC/C^Cdh1 on the maintenance of genome integrity and show that this is, at least partially, achieved by controlling CtIP stability in a cell cycle‐ and DNA damage‐dependent manner.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.