Raimundo Freire

Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.

Polo-like Kinase-1 Controls Proteasome-Dependent Degradation of Claspin during Checkpoint Recovery

DNA-damage checkpoints maintain genomic integrity by mediating a cell-cycle delay in response to genotoxic stress or stalled replication forks. In response to damage, the checkpoint kinase ATR phosphorylates and activates its effector kinase Chk1 in a process that critically depends on Claspin . However, it is not known how exactly this kinase cascade is silenced. Here we demonstrate that the abundance of Claspin is regulated through proteasomal degradation. In response to DNA damage, Claspin is transiently stabilized, and its expression depends on Chk1 kinase activity. In addition, we show that Claspin is degraded upon mitotic entry, a process that depends on the beta-TrCP-SCF ubiquitin ligase and Polo-like kinase-1 (Plk1). We demonstrate that Claspin interacts with both beta-TrCP and Plk1 and that inactivation of these components or the beta-TrCP recognition motif in Claspin prevents its mitotic degradation. Interestingly, expression of a nondegradable Claspin mutant inhibits recovery from a DNA-damage-induced checkpoint arrest. Thus, we conclude that Claspin levels are tightly regulated, both during unperturbed cell cycles and after DNA damage. Moreover, our data demonstrate that the degradation of Claspin at the onset of mitosis is an essential step for the recovery of a cell from a DNA-damage-induced cell-cycle arrest.

The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability

Rothmund-Thomson syndrome (RTS) is a human genetic disorder characterized by genome instability, cancer susceptibility and premature aging. The gene defective in a subset of RTS cases, RECQL4, encodes a member of the RecQ family of DNA helicases. To better define the function of the RECQL4 protein, we have determined its subcellular localization. We have raised antibodies against the N- and C-terminal parts of RECQL4 and could show that in various human cells endogenous RECQL4 forms discrete nuclear foci that colocalize with promyelotic leukaemia protein (PML). The number of foci and their colocalization with PML does not significantly change after induction of different types of DNA damages. Silencing of RECQL4 expression by siRNA causes a significant reduction in RECQL4 nuclear foci formation. Furthermore, we demonstrate that RECQL4 foci coincide with foci formed by human Rad51 and regions of single-stranded DNA after induction of DNA double-strand breaks. In agreement with this, we also show that RECQL4 and Rad51 form a complex in human cells. Our findings suggest a role for RECQL4 in the repair of DNA double-strand breaks by homologous recombination and shed new light onto RECQL4s function in human cells.

The association of ATR protein with mouse meiotic chromosome cores

Abstract. The ATR (ataxia telangiectasia- and RAD3-related) protein is present on meiotic prophase chromosome cores and paired cores (synaptonemal complexes, SCs). Its striking characteristic is that the protein forms dense aggregates on the cores and SCs of the last chromosomes to pair at the zygotene-pachytene transition. It would appear that the ATR protein either signals delays in pairing or it is directly involved in the completion of the pairing phase. Atm-deficient spermatocytes, which are defective in the chromosome pairing phase, accumulate large amounts of ATR. The behaviour of ATR at meiotic prophase sets it apart from the distribution of the RAD51/DMC1 recombinase complex and our electron microscope observations confirm that they do not co-localize. We failed to detect ATM in association with cores/SCs and we have reported elsewhere that RAD1 protein does not co-localize with DMC1 foci. The expectation that putative DNA-damage checkpoint proteins, ATR, ATM and RAD1, are associated with RAD51/DMC1 recombination sites where DNA breaks are expected to be present, is therefore not supported by our observations.

Simian Virus 40 Large T Antigen Disrupts Genome Integrity and Activates a DNA Damage Response via Bub1 Binding

ABSTRACT Simian virus 40 (SV40) large T antigen (LT) is a multifunctional protein that is important for viral replication and oncogenic transformation. Previously, infection of monkey or human cells with SV40 was shown to lead to the induction of DNA damage response signaling, which is required for efficient viral replication. However, it was not clear if LT is sufficient to induce the damage response and, if so, what the genetic requirements and functional consequences might be. Here, we show that the expression of LT alone, without a replication origin, can induce key DNA damage response markers including the accumulation of γ-H2AX and 53BP1 in nuclear foci. Other DNA damage-signaling components downstream of ATM/ATR kinases were induced, including chk1 and chk2. LT also bound the Claspin mediator protein, which normally facilitates the ATR activation of chk1 and monitors cellular replication origins. Stimulation of the damage response by LT depends mainly on binding to Bub1 rather than to the retinoblastoma protein. LT has long been known to stabilize p53 despite functionally inactivating it. We show that the activation of a DNA damage response by LT via Bub1 appears to play a major role in p53 stabilization by promoting the phosphorylation of p53 at Ser15. Accompanying the DNA damage response, LT induces tetraploidy, which is also dependent on Bub1 binding. Taken together, our data suggest that LT, via Bub1 binding, breaches genome integrity mechanisms, leading to DNA damage responses, p53 stabilization, and tetraploidy.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

Co-operation of BRCA1 and POH1 relieves the barriers posed by 53BP1 and RAP80 to resection

In G2 phase cells, DNA double-strand break repair switches from DNA non-homologous end-joining to homologous recombination. This switch demands the promotion of resection. We examine the changes in 53BP1 and RAP80 ionizing radiation induced foci (IRIF) in G2 phase, as these are factors that restrict resection. We observed a 2-fold increase in the volume of 53BP1 foci by 8 h, which is not seen in G1 cells. Additionally, an IRIF core devoid of 53BP1 arises where RPA foci form, with BRCA1 IRIF forming between 53BP1 and replication protein A (RPA). Ubiquitin chains assessed using α-FK2 antibodies are similarly repositioned. Repositioning of all these components requires BRCA1’s BRCT but not the ring finger domain. 53BP1, RAP80 and ubiquitin chains are enlarged following POH1 depletion by small interfering RNA, but a devoid core does not form and RPA foci formation is impaired. Co-depletion of POH1 and RAP80, BRCC36 or ABRAXAS allows establishment of the 53BP1 and ubiquitin chain-devoid core. Thus, the barriers posed by 53BP1 and RAP80 are relieved by BRCA1 and POH1, respectively. Analysis of combined depletions shows that these represent distinct but interfacing barriers to promote loss of ubiquitin chains in the IRIF core, which is required for subsequent resection. We propose a model whereby BRCA1 impacts on 53BP1 to allow access of POH1 to RAP80. POH1-dependent removal of RAP80 within the IRIF core enables degradation of ubiquitin chains, which promotes loss of 53BP1. Thus, POH1 represents a novel component regulating the switch from non-homologous end-joining to homologous recombination.

The Forkhead Box M1 protein regulates BRIP1 expression and DNA damage repair in epirubicin treatment

FOXM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Here, we establish that γH2AX foci, indicative of DNA double-strand breaks (DSBs), accumulate in a time-dependent manner in the drug-sensitive MCF-7 cells but not in the resistant counterparts in response to epirubicin. We find that FOXM1 expression is associated with epirubicin sensitivity and DSB repair. Ectopic expression of FOXM1 can increase cell viability and abrogate DSBs sustained by MCF-7 cells following epirubicin, owing to an enhancement in repair efficiency. Conversely, alkaline comet and γH2AX foci formation assays show that Foxm1-null cells are hypersensitive to DNA damage, epirubicin and γ-irradiation. Furthermore, we find that FOXM1 is required for DNA repair by homologous recombination (HR) but not non-homologous end joining (NHEJ), using HeLa cell lines harbouring an integrated direct repeat green fluorescent protein reporter for DSB repair. We also identify BRIP1 as a direct transcription target of FOXM1 by promoter analysis and chromatin-immunoprecipitation assay. In agreement, depletion of FOXM1 expression by small interfering RNA downregulates BRIP1 expression at the protein and mRNA levels in MCF-7 and the epirubicin-resistant MCF-7 EpiR cells. Remarkably, the requirement for FOXM1 for DSB repair can be circumvented by reintroduction of BRIP1, suggesting that BRIP1 is an important target of FOXM1 in DSB repair. Indeed, like FOXM1, BRIP1 is needed for HR. These data suggest that FOXM1 regulates BRIP1 expression to modulate epirubicin-induced DNA damage repair and drug resistance.

FOXM1 targets NBS1 to regulate DNA damage-induced senescence and epirubicin resistance.

FOXM1 is implicated in genotoxic drug resistance but its mechanism of action remains elusive. We show here that FOXM1-depletion can sensitize breast cancer cells and mouse embryonic fibroblasts (MEFs) into entering epirubicin-induced senescence, with the loss of long-term cell proliferation ability, the accumulation of γH2AX foci, and the induction of senescence-associated β-galactosidase activity and cell morphology. Conversely, reconstitution of FOXM1 in FOXM1-deficient MEFs alleviates the accumulation of senescence-associated γH2AX foci. We also demonstrate that FOXM1 regulates NBS1 at the transcriptional level through an forkhead response element on its promoter. Like FOXM1, NBS1 is overexpressed in the epirubicin-resistant MCF-7EpiR cells and its expression level is low but inducible by epirubicin in MCF-7 cells. Consistently, overexpression of FOXM1 augmented and FOXM1 depletion reduced NBS1 expression and epirubicin-induced ataxia-telangiectasia mutated (ATM)phosphorylation in breast cancer cells. Together these findings suggest that FOXM1 increases NBS1 expression and ATM phosphorylation, possibly through increasing the levels of the MRN(MRE11/RAD50/NBS1) complex. Consistent with this idea, the loss of P-ATM induction by epirubicin in the NBS1-deficient NBS1-LBI fibroblasts can be rescued by NBS1 reconstitution. Resembling FOXM1, NBS1 depletion also rendered MCF-7 and MCF-7EpiR cells more sensitive to epirubicin-induced cellular senescence. In agreement, the DNA repair-defective and senescence phenotypes in FOXM1-deficent cells can be effectively rescued by overexpression of NBS1. Moreover, overexpression of NBS1 and FOXM1 similarly enhanced and their depletion downregulated homologous recombination (HR) DNA repair activity. Crucially, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, demonstrating that NBS1 is indispensable for the HR function of FOXM1. The physiological relevance of the regulation of NBS1 expression by FOXM1 is further underscored by the strong and significant correlation between nuclear FOXM1 and total NBS1 expression in breast cancer patient samples, further suggesting that NBS1 as a key FOXM1 target gene involved in DNA damage response, genotoxic drug resistance and DNA damage-induced senescence.

Bora and Aurora-A continue to activate Plk1 in mitosis

ABSTRACT Polo-like kinase-1 (Plk1) is required for proper cell division. Activation of Plk1 requires phosphorylation on a conserved threonine in the T-loop of the kinase domain (T210). Plk1 is first phosphorylated on T210 in G2 phase by the kinase Aurora-A, in concert with its cofactor Bora. However, Bora was shown to be degraded prior to entry into mitosis, and it is currently unclear how Plk1 activity is sustained in mitosis. Here we show that the Bora–Aurora-A complex remains the major activator of Plk1 in mitosis. We show that a small amount of Aurora-A activity is sufficient to phosphorylate and activate Plk1 in mitosis. In addition, a fraction of Bora is retained in mitosis, which is essential for continued Aurora-A-dependent T210 phosphorylation of Plk1. We find that once Plk1 is activated, minimal amounts of the Bora–Aurora-A complex are sufficient to sustain Plk1 activity. Thus, the activation of Plk1 by Aurora-A may function as a bistable switch; highly sensitive to inhibition of Aurora-A in its initial activation, but refractory to fluctuations in Aurora-A activity once Plk1 is fully activated. This provides a cell with robust Plk1 activity once it has committed to mitosis.