Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase (original) (raw)
Related papers
Molecular Biology of the Cell, 1999
Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLMencodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of aSaccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern.
Phosphorylation of the Bloom's Syndrome Helicase and Its Role in Recovery from S-Phase Arrest
Molecular and Cellular Biology, 2004
Bloom's syndrome (BS) is a human genetic disorder associated with cancer predisposition. The BS gene product, BLM, is a member of the RecQ helicase family, which is required for the maintenance of genome stability in all organisms. In budding and fission yeasts, loss of RecQ helicase function confers sensitivity to inhibitors of DNA replication, such as hydroxyurea (HU), by failure to execute normal cell cycle progression following recovery from such an S-phase arrest. We have examined the role of the human BLM protein in recovery from S-phase arrest mediated by HU and have probed whether the stress-activated ATR kinase, which functions in checkpoint signaling during S-phase arrest, plays a role in the regulation of BLM function. We show that, consistent with a role for BLM in protection of human cells against the toxicity associated with arrest of DNA replication, BS cells are hypersensitive to HU. BLM physically associates with ATR (ataxia telangiectasia and rad3 + related) pr...
Bloom helicase is involved in DNA surveillance in early S phase in vertebrate cells
Oncogene, 2001
Bloom syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and an elevated risk of malignancy. The gene mutated in BS, BLM, encodes a RecQ-type DNA helicase. BS cells have mutator phenotypes such as hyper-recombination, chromosome instability and an increased frequency of sister chromatid exchange (SCE). To define the primary role of BLM, we generated BLM(-/-) mutants of the chicken B-cell line DT40. In addition to characteristics of BLM(-/-) cells reported previously by the other group, they are hypersensitive to genotoxic agents such as etoposide, bleomycin and 4-nitroquinoline-1-oxide and irradiation with the short wave length of UV (UVC) light, whereas they exhibit normal sensitivity to X-ray irradiation and hydroxyurea. UVC irradiation to BLM(-/-) cells during G(1) to early S phase caused chromosomal instability such as chromatid breaks and chromosomal quadriradials, leading to eventual cell death. These results suggest that BLM i...
Journal of Biological Chemistry, 2002
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.
Oncogene, 1998
Bloom's syndrome (BS) is a rare human genetic disorder characterized by mutations within the BLM gene whose primary eects are excessive chromosome breakaoge and increased rates of sister chromatide interchange in somatic cells. We report the characterization of a murine protein (mBLM), highly related to the product of the human BLM gene. This protein exhibits an ATPdependent DNA-helicase activity that unwinds DNA in a 3' ± 5' direction. Single aminoacid substitutions found in BS cells, abolish both ATPase and helicase activities of this protein, indicating that defects in these BLM functions may be primarily responsible for BS establishment. These results provide the ®rst evidence suggesting that the enzymatic activities of the BLM product are implicated in the upholding of genomic integrity.
Oncogene, 2015
Mutations in Bloom helicase (BLM) lead to Bloom Syndrome (BS). BS is characterized by multiple clinical manifestations including predisposition to a wide spectrum of cancers. Studies have revealed the mechanism of BLM recruitment after stalled replication and its role during the repair of DNA damage. We now provide evidence that BLM undergoes K48-linked ubiquitylation and subsequent degradation during mitosis due to the E3 ligase, Fbw7α. Fbw7α carries out its function after GSK3β- and CDK2/cyclin A2-dependent phosphorylation events on Thr171 and Ser175 of BLM which lies within a well-defined phosphodegron, a sequence which is conserved in all primates. Phosphorylation on BLM Thr171 and Ser175 depends on prior phosphorylation at Thr182 by Chk1/Chk2. Thr182 phosphorylation not only controls BLM ubiquitylation and degradation during mitosis but is also a determinant for its localization on the ultrafine bridges. Consequently lack of Thr182 phosphorylation leads to multiple manifestatio...
Nucleic Acids Research, 2008
Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL d (hPOL d). The hPOL d enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL d strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL d. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL d.
Possible anti-recombinogenic role of Bloom's syndrome helicase in double-strand break processing
Nucleic Acids Research, 2003
Bloom's syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3¢±5¢ DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA doublestrand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report ®rst that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcsdependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, re¯ecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.
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.