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By necessity, the ancient activity of type II topoisomerases co-evolved with the double-helical structure of DNA, at least in organisms with circular genomes. In humans, the strand passage reaction of DNA topoisomerase II (Topo II) is the target of several major classes of cancer drugs which both poison Topo II and activate cell cycle checkpoint controls. It is important to know the cellular effects of molecules that target Topo II, but the mechanisms of checkpoint activation that respond to Topo II dysfunction are not well understood. Here, we provide evidence that a checkpoint mechanism monitors the strand passage reaction of Topo II. In contrast, cells do not become checkpoint arrested in the presence of the aberrant DNA topologies, such as hyper-catenation, that arise in the absence of Topo II activity. An overall reduction in Topo II activity (i.e. slow strand passage cycles) does not activate the checkpoint, but specific defects in the T-segment transit step of the strand passage reaction do induce a cell cycle delay. Furthermore, the cell cycle delay depends on the divergent and catalytically inert C-terminal region of Topo II, indicating that transmission of a checkpoint signal may occur via the C-terminus. Other, well characterized, mitotic checkpoints detect DNA lesions or monitor unattached kinetochores; these defects arise via failures in a variety of cell processes. In contrast, we have described the first example of a distinct category of checkpoint mechanism that monitors the catalytic cycle of a single specific enzyme in order to determine when chromosome segregation can proceed faithfully.
Oncogene-induced senescence can provide a protective mechanism against tumour progression. However, production of cytokines and growth factors by senescent cells may contribute to tumour development. Thus, it is unclear whether induction of senescence represents a viable therapeutic approach. Here, using a mouse model with orthotopic implantation of metastatic melanoma tumours taken from 19 patients, we observed that targeting aurora kinases with MLN8054/MLN8237 impaired mitosis, induced senescence and markedly blocked proliferation in patient tumour implants. Importantly, when a subset of tumour-bearing mice were monitored for tumour progression after pausing MLN8054 treatment, 50% of the tumours did not progress over a 12-month period. Mechanistic analyses revealed that inhibition of aurora kinases induced polyploidy and the ATM/Chk2 DNA damage response, which mediated senescence and a NF-κB-related, senescence-associated secretory phenotype (SASP). Blockade of IKKβ/NF-κB led to reversal of MLN8237-induced senescence and SASP. Results demonstrate that removal of senescent tumour cells by infiltrating myeloid cells is crucial for inhibition of tumour re-growth. Altogether, these data demonstrate that induction of senescence, coupled with immune surveillance, can limit melanoma growth.
Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.
BACKGROUND - More than 1000 reports have been published in the past two decades on associations between variants in candidate genes and risk of breast cancer. Results have been generally inconsistent. We did a literature search and meta-analyses to provide a synopsis of the current understanding of the genetic architecture of breast-cancer risk.
METHODS - A systematic literature search for candidate-gene association studies of breast-cancer risk was done in two stages, using PubMed on or before Feb 28, 2010. A total of 24,500 publications were identified, of which 1059 were deemed eligible for inclusion. Meta-analyses were done for 279 genetic variants in 128 candidate genes or chromosomal loci that had at least three data sources. Variants with significant associations by meta-analysis were assessed using the Venice criteria and scored as having strong, moderate, or weak cumulative evidence for an association with breast-cancer risk.
FINDINGS - 51 variants in 40 genes showed significant associations with breast-cancer risk. Cumulative epidemiological evidence of an association was graded as strong for ten variants in six genes (ATM, CASP8, CHEK2, CTLA4, NBN, and TP53), moderate for four variants in four genes (ATM, CYP19A1, TERT, and XRCC3), and weak for 37 variants. Additionally, in meta-analyses that included a minimum of 10,000 cases and 10,000 controls, convincing evidence of no association with breast-cancer risk was identified for 45 variants in 37 genes.
INTERPRETATION - Whereas most genetic variants assessed in previous candidate-gene studies showed no association with breast-cancer risk in meta-analyses, 14 variants in nine genes had moderate to strong evidence for an association. Further evaluation of these variants is warranted.
FUNDING - US National Cancer Institute.
Copyright © 2011 Elsevier Ltd. All rights reserved.
In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named ;no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis.
The Chk2 protein kinase protects genome integrity by promoting cell cycle arrest or apoptosis in response to DNA double-strand breaks, and Chk2 mutations are found in both familial and sporadic cancers. Exposure of cells to ionizing radiation (IR) or radiomimetic drugs induces Chk2 phosphorylation by ATM, followed by Chk2 oligomerization, auto-/transphosphorylation, and activation. Here we demonstrate that Chk2 is ubiquitinated upon activation and that this requires Chk2 kinase activity. Serine 379 (S379) was identified as a novel IR-inducible autophosphorylation site required for ubiquitination of Chk2 by a Cullin 1-containing E3 ligase complex. Importantly, S379 was required for Chk2 to induce apoptosis in cells with DNA double-strand breaks. Thus, auto-/transphosphorylation of S379 is required for Chk2 ubiquitination and effector function.
We describe a method for the analysis of multi-site phosphorylation in serine/threonine (Ser/Thr)-rich protein sequences. Site-specific mutagenesis was used to introduce tryptic cleavage sites in the serine glutamine/threonine glutamine cluster domain (SCD) of the human checkpoint protein kinase (Chk2). The mutant proteins were shown to autophosphorylate on residues that are inducibly phosphorylated when mammalian cells are exposed to ionizing radiation (serine 33/35, serine 516, threonine 68 and threonine 432). Five Ser/Thr clusters within the SCD were flanked by arginine or lysine residues to produce tryptic peptides for nanospray liquid chromatography (nanoLC)/linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry. Phosphorylation sites were assigned using accurate-mass-driven analysis and interpretation of low-energy collision-induced dissociation spectra acquired in the ion trap. In addition to verifying known phosphorylation sites, seventeen novel sites were identified within the SCD of Chk2. The approach should be applicable to other O-linked post-translational modifications that occur in proteins with Ser/Thr-rich sequences.
Copyright (c) 2007 John Wiley & Sons, Ltd.
Mutation of human microcephalin (MCPH1) causes autosomal recessive primary microcephaly, a developmental disorder characterized by reduced brain size. We identified mcph1, the Drosophila homolog of MCPH1, in a genetic screen for regulators of S-M cycles in the early embryo. Embryos of null mcph1 female flies undergo mitotic arrest with barrel-shaped spindles lacking centrosomes. Mutation of Chk2 suppresses these defects, indicating that they occur secondary to a previously described Chk2-mediated response to mitotic entry with unreplicated or damaged DNA. mcph1 embryos exhibit genomic instability as evidenced by frequent chromatin bridging in anaphase. In contrast to studies of human MCPH1, the ATR/Chk1-mediated DNA checkpoint is intact in Drosophila mcph1 mutants. Components of this checkpoint, however, appear to cooperate with MCPH1 to regulate embryonic cell cycles in a manner independent of Cdk1 phosphorylation. We propose a model in which MCPH1 coordinates the S-M transition in fly embryos: in the absence of mcph1, premature chromosome condensation results in mitotic entry with unreplicated DNA, genomic instability, and Chk2-mediated mitotic arrest. Finally, brains of mcph1 adult male flies have defects in mushroom body structure, suggesting an evolutionarily conserved role for MCPH1 in brain development.
DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.
G1-specific transcription in yeast depends upon SBF and MBF. We have identified Nrm1 (negative regulator of MBF targets 1), as a stable component of MBF. NRM1 (YNR009w), an MBF-regulated gene expressed during late G1 phase, associates with G1-specific promoters via MBF. Transcriptional repression upon exit from G1 phase requires both Nrm1 and MBF. Inactivation of Nrm1 results in prolonged expression of MBF-regulated transcripts and leads to hydroxyurea (HU) resistance and enhanced bypass of rad53Delta- and mec1Delta-associated lethality. Constitutive expression of a stabilized form of Nrm1 represses MBF targets and leads to HU sensitivity. The fission yeast homolog SpNrm1, encoded by the MBF target gene nrm1(+) (SPBC16A3.07c), binds to MBF target genes and acts as a corepressor. In both yeasts, MBF represses G1-specific transcription outside of G1 phase. A negative feedback loop involving Nrm1 bound to MBF leads to transcriptional repression as cells exit G1 phase.
We describe the data-dependent analysis of protein phosphorylation using rapid-acquisition nano-LC-linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FTMS). The accurate m/z values of singly, doubly, and triply charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr, or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FTMS and collision-induced dissociation spectra, 11 novel Chk2 autophosphorylation sites were assigned. Additionally, the presence of additional Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues.