Dephosphorylation and Subcellular Compartment Change of the Mitotic Bloom's Syndrome DNA Helicase in Response to Ionizing Radiation

Abstract

Bloom's syndrome is a rare human autosomal recessive disorder that combines a marked genetic instability and an increased risk of developing all types of cancers and which results from mutations in both copies of the BLM gene encoding a RecQ 3 -5 DNA helicase. We recently showed that BLM is phosphorylated and excluded from the nuclear matrix during mitosis. We now show that the phosphorylated mitotic BLM protein is associated with a 3 -5 DNA helicase activity and interacts with topoisomerase III␣. We demonstrate that in mitosis-arrested cells, ionizing radiation and roscovitine treatment both result in the reversion of BLM phosphorylation, suggesting that BLM could be dephosphorylated through the inhibition of cdc2 kinase. This was supported further by our data showing that cdc2 kinase activity is inhibited in ␥-irradiated mitotic cells. Finally we show that after ionizing radiation, BLM is not involved in the establishment of the mitotic DNA damage checkpoint but is subjected to a subcellular compartment change. These findings lead us to propose that BLM may be phosphorylated during mitosis, probably through the cdc2 pathway, to form a pool of rapidly available active protein. Inhibition of cdc2 kinase after ionizing radiation would lead to BLM dephosphorylation and possibly to BLM recruitment to some specific sites for repair.Mutations in both copies of the BLM gene give rise to Bloom's syndrome (BS), 1 a rare disorder characterized by marked genetic instability combined with a greatly increased predisposition to a wide range of cancers commonly affecting the general population. The BLM gene is located on chromosome 15 at 15q26.1 and encodes the BLM protein, which belongs to the DExH box-containing RecQ helicase subfamily (1) and displays ATP-and Mg 2ϩ -dependent 3Ј-5Ј-DNA helicase activity (2). The major cellular consequences of a BLM defect are an increase in homologous recombination and in the rate of widespread mutations. Indeed, BS cells display spontaneous hypermutability and several cytogenetic abnormalities including an increase in chromosome breaks, symmetric quadriradial chromatid interchanges between homologous chromosomes, and sister chromatid exchanges (for review, see Ref.3). Until recently, very little information was available about the physiological function of BLM. Now, several lines of evidences strongly support the involvement of BLM during DNA replication and in the cellular response to DNA damage. Recently, BLM protein has been shown to accumulate during the S phase of the cell cycle (4), to interact selectively in vitro with Holliday junctions (5), and to coimmunoprecipitate with hRAD51 from cells synchronized in early S phase (6). BLM has also been shown to participate in the BRCA1-associated genome surveillance complex (7), to be phosphorylated and to accumulate through an ATM-dependent pathway in response to ionizing radiation (8), to assemble with promyelocytic leukemia protein at sites of single-stranded DNA after ␥-irradiation (9), and to be cleaved early during apoptosis (10).We s...

The Bloom Syndrome Helicase is a Substrate of the Mitotic Cdc2 Kinase

“…The EBV-transformed lymphoblastoid B cell lines D1 (control) and GM03189B (AT), and HeLa cells were cultured as previously described. 5,9 Chemicals. Demecolcine, nocodazole and roscovitine (Sigma) were resuspended in dimethylsulfoxide (DMSO) to give stock solutions at concentrations of 26 mM, 0.2 mg/ml and 75 mM, respectively.…”

“…4 However, mitotic BLM phosphorylation affects neither 3'-5' DNA helicase activity nor interaction with topoisomerase IIIα. 5 Both BLM phosphorylation and exclusion from the nuclear matrix are abolished by the inactivation of cdc2 kinase by either the introduction of numerous DNA double-strand breaks or the cdc2 kinase inhibitor roscovitine, 5,6 suggesting that BLM may be directly phosphorylated by the cdc2 kinase during mitosis. 6 However, mitotic phosphorylation of BLM is also partly dependent on ATM kinase, 7 and ATM phosphorylation sites have been identified at Thr-99 and Thr-122 in BLM.…”

A Major Role for Mitotic cdc2 Kinase Inactivation in the Establishment of the Mitotic DNA Damage Checkpoint

“…DNA damage during the spindle assembly checkpoint prevents exit from mitosis (3,19). To determine whether the activation of the mitotic checkpoint in response to DNA damage depends on cdc2 activity, we analyzed the effect of TSA, CLQ, and roscovitine on exit from mitosis.…”

“…BLM is phosphorylated during mitosis (2), and reversion of this phosphorylation is associated with inactivation of cdc2 kinase (3). Mitotic surveillance mechanisms include stabilization and inactivation of cdc2 kinase.…”

Syndrome de Bloom : hétérozygotie et prédisposition au cancer

“…Ces résultats ont été reproduits par l'équipe de Joanna Groden dans un modèle murin [2]. En effet, cette équipe a développé des souris chez lesquelles une copie de l'homologue murin du gène BLM a été invalidée, produisant ainsi un allèle mutant Blm Cin [7] dans des mécanismes de surveillance du génome [8], de réso-lution de structures anormales de l'ADN [9,10], et/ou de réparation de lésions de l'ADN, notamment en réponse à des stress génotoxiques [11,12]. Si la protéine BLM est impliquée dans de tels processus au sein de complexes protéiques, et dans le cadre d'interactions stoechiométriques bien déterminées, alors une réduction de la quantité de protéine BLM pourrait être délétère pour la cellule.…”