Dephosphorylation and Subcellular Compartment Change of the Mitotic Bloom's Syndrome DNA Helicase in Response to Ionizing Radiation (original) (raw)
Related papers
Oncogene, 2000
Bloom's syndrome (BS), a rare genetic disease, arises through mutations in both alleles of the BLM gene which encodes a 3' ± 5' DNA helicase identi®ed as a member of the RecQ family. BS patients exhibit a high predisposition to development of all types of cancer aecting the general population and BLM-de®cient cells display a strong genetic instability. We recently showed that BLM protein expression is regulated during the cell cycle, accumulating to high levels in S phase, persisting in G2/ M and sharply declining in G1, suggesting a possible implication of BLM in a replication (S phase) and/or post-replication (G2 phase) process. Here we show that, in response to ionizing radiation, BLM-de®cient cells exhibit a normal p53 response as well as an intact G1/S cell cycle checkpoint, which indicates that ATM and p53 pathways are functional in BS cells. We also show that the BLM defect is associated with a partial escape of cells from the g-irradiation-induced G2/M cell cycle checkpoint. Finally, we present data demonstrating that, in response to ionizing radiation, BLM protein is phosphorylated and accumulates through an ATMdependent pathway. Altogether, our data indicate that BLM participates in the cellular response to ionizing radiation by acting as an ATM kinase downstream eector.
Mutation Research/Reviews in Mutation Research, 2012
The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. ''Superfluous'' protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.
Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase
Oncogene, 2000
Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' ± 5' DNA helicase identi®ed as a member of the RecQ family. We developed polyclonal antibodies speci®c for the NH 2 -and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/ M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modi®cations in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process.
Dna Repair, 2008
Double strand breaks (DSB) are critical lesions involved in the formation of chromosomal aberrations. In response to DNA damage, the cell has mechanisms of repair and cell-cycle control to maintain the genome integrity in which BRCA1 gene is implicated. In the present study an evaluation of the radio-induced damage in G 2 phase of the cell cycle in lymphocytes from BRCA1 heterozygotes is presented. For this purpose Calyculin-A-based premature chromosome condensation (PCC) combined with mitotic arrest has been applied to examine with conventional cytogenetics the damage in G 2 and M phase cells, and to evaluate the G 2 -to-M phase transition. Irradiated peripheral blood lymphocytes from seven heterozygote females (BRCA1 +/− ) and seven control females (BRCA1 +/+ ) have been analyzed. The mean proportion of G 2 cells in BRCA1 +/− was significantly higher than in BRCA1 +/+ , indicating a higher G 2 delay after IR exposure in cells from BRCA1 +/− females. On the other hand, whereas the mean frequency of chromatid breaks (chtb) in G 2 cells was not statistically different between both groups, the mean frequency of chtb in M cells of the BRCA1 +/− group was significantly higher than in the BRCA1 +/+ one. Moreover, the mean proportion of M cells with aberrations was significantly higher in BRCA1 +/− than in BRCA1 +/+ suggesting that in spite of the higher G 2 delay of BRCA1 +/− more damaged cells are able to pass the G 2 -to-M transition. (J. Francesc Barquinero). gen species. IR induces base damage (BD), and single-and double-strand breaks (SSB, DSB). The cell has complex signal transduction, cell-cycle checkpoints and repair pathways to respond to the DNA damage. BD and SSB are repaired by 1568-7864/$ -see front matter
Evasion of Early Cellular Response Mechanisms following Low Level Radiation-induced DNA Damage
Journal of Biological Chemistry, 2004
DNA damage that is not repaired with high fidelity can lead to chromosomal aberrations or mitotic cell death. To date, it is unclear what factors control the ultimate fate of a cell receiving low levels of DNA damage (i.e. survival at the risk of increased mutation or cell death). We investigated whether DNA damage could be introduced into human cells at a level and frequency that could evade detection by cellular sensors of DNA damage. To achieve this, we exposed cells to equivalent doses of ionizing radiation delivered at either a high dose rate (HDR) or a continuous low dose rate (LDR). We observed reduced activation of the DNA damage sensor ataxia-telangiectasia mutated (ATM) and its downstream target histone H2A variant (H2AX) following LDR compared with HDR exposures in both cancerous and normal human cells. This lack of DNA damage signaling was associated with increased amounts of cell killing following LDR exposures. Increased killing by LDR radiation has been previously termed the "inverse dose rate effect," an effect for which no clear molecular processes have been described. These LDR effects could be abrogated by the preactivation of ATM or simulated in HDR-treated cells by inhibiting ATM function. These data are the first to demonstrate that DNA damage introduced at a reduced rate does not activate the DNA damage sensor ATM and that failure to activate ATMassociated repair pathways contributes to the increased lethality of continuous LDR radiation exposures. This inactivation may reflect one strategy by which cells avoid accumulating mutations as a result of error-prone DNA repair and may have a broad range of implications for carcinogenesis and, potentially, the clinical treatment of solid tumors.
DNA damage in cells exhibiting radiation-induced genomic instability
Mutagenesis, 2015
Cells exhibiting radiation-induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesis that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated ...
Carcinogenesis, 2012
The vertebrate RECQL4 (RECQ4) gene is thought to be the ortholog of budding yeast SLD2. However, RecQL4 contains within its C-terminus a RecQ-like helicase domain, which is absent in Sld2. We established human pre-B lymphocyte Nalm-6 cells, in which the endogenous RECQL4 gene was homozygously targeted such that the entire C-terminus would not be expressed. The RECQL4(DC/DC) cells behaved like the parental cells during unperturbed DNA replication or after treatment with agents that induce stalling of DNA replication forks, such as hydroxyurea (HU). However, after exposure to ionizing radiation (IR), the RECQL4(DC/DC) cells exhibited hypersensitivity, inability to complete S phase and prematurely terminated or paused DNA replication forks. Deletion of BLM, a gene that also encodes a RecQ helicase, had the opposite phenotype; an almost wild-type response to IR, but hypersensitivity to HU. Targeting both RECQL4 and BLM resulted in viable cells, which exhibited mostly additive phenotypes compared with those exhibited by the RECQL4(DC/DC) and the BLM(2/2) cells. We propose that RecQL4 facilitates DNA replication in cells that have been exposed to IR.
Cancer Research
I In 1KOBRcell line was derived from an acute Ivmphohlastic leukemia patient »hooverresponded to radiation therapy and died following radi ation morbidity. ISill! U cells are hypersensitive to the lethal effects of ionizing radiation and are defective in the repair of DNA double-strand breaks (DSBs). The levels and activity of the proteins of the DNA-dependent protein kinase complex are normal in IXOBR cells. To facilitate a measurement of Ml)).| recombination, we have characterized ISOI'.KM. a SV40-transformed line derived from ISOIiK. ISilliKM retains the radiosensitivity and defect in DSU repair characteristic of ISill!U. The activities associated with DNA-dependent protein kinase are also normal in 1SOKKM cells. The ability to carry out V(D)J recombination is compara ble in I80BKM and a reference control transformed human cell line. MRC5V1. These results show that I80BK and ISIHSKM differ from the rodent mutants belonging to ionizing radiation complementation groups 4, 5, 6, and 7 and, therefore, represent a new mutant phenotype, in which a delect in DNA DSB rejoining is not associated with defective V(D)J recombination. Furthermore, we have shown that ISOUK can arrest at the G,-S and (..-M cell cycle checkpoints after irradiation. These results confirm that 1SOBR can be distinguished from ataxia telangiectasia.
Journal of Molecular Biology, 2008
Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ATR kinase to induce transient double stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase α from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.