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ABSTRACT: Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair.
Nature Structural & Molecular Biology 12/2007; 14(11):1096-104. · 12.71 Impact Factor
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ABSTRACT: The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.
Molecular Cancer Research 08/2007; 5(7):713-24. · 4.29 Impact Factor
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ABSTRACT: Mutations in BLM give rise to Bloom's syndrome, a genetic disorder associated with cancer predisposition and chromosomal instability. Using a dual-labeling system in isolated chromosome fibers, we show that the BLM protein is required for two aspects of the cellular response to replicative stress: efficient replication-fork restart and suppression of new origin firing. These functions require the helicase activity of BLM and the Thr99 residue targeted by stress-activated kinases.
Nature Structural & Molecular Biology 08/2007; 14(7):677-9. · 12.71 Impact Factor
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ABSTRACT: 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) protein and is phosphorylated on two residues in the N-terminal domain, Thr-99 and Thr-122, by this kinase. Moreover, BS cells ectopically expressing a BLM protein containing phosphorylation-resistant T99A/T122A substitutions fail to adequately recover from an HU-induced replication blockade, and the cells subsequently arrest at a caffeine-sensitive G(2)/M checkpoint. These abnormalities are not associated with a failure of the BLM-T99A/T122A protein to localize to replication foci or to colocalize either with ATR itself or with other proteins that are required for response to DNA damage, such as phosphorylated histone H2AX and RAD51. Our data indicate that RecQ helicases play a conserved role in recovery from perturbations in DNA replication and are consistent with a model in which RecQ helicases act to restore productive DNA replication following S-phase arrest and hence prevent subsequent genomic instability.
Molecular and Cellular Biology 03/2004; 24(3):1279-91. · 5.53 Impact Factor
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ABSTRACT: Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designatec topoisomerase IIα (170 kDa form) and topoisomerase IIβ (180 kDa form). We have isolated cDNA clones encoding the β isozyme from a human B-cell library. The proposed coding region for the topoisomerase IIβ protein is 4,863 nucleotides long and would encode i polypeptide with a calculated M, of 182,705. The predicted topoisomerase IIβ protein sequence show; striking similarity (72% identical residues) to that of the human α isozyme, and homology to topoisomerase II proteins from Drosophila , yeast and bacteria. Region of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising −25% of the total protein. We have quantified the level of topoisomerase IIβ mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase IIα mRNA. Topoisomerase IIβ mRNA was expressed inhaemopoietic, epithelial and fibroblast cell lines, although of different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase IIα and β genes. We have localised the gene encoding topoisomerase IIβ protein to chromosome 3p24 in the human genome.
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ABSTRACT: Screening of a human B-cell cDNA library with a topoisomerase 11/3 gene-specific probe revealed the presence of two distinct forms of topoisomerase 11/3 cDNA. One form (designated topoisomerase 11/3-1), representing the majority of the clones, would encode the topoisomerase 11/3 amino acid sequence reported recently [Jenkins, J.R. et al . (1992) Nucleic Acids Res ., 20, 5587-5592]. The second form (designated topoisomerase IIβ-2) would encode a protein containing an additional 5 amino acids inserted after Vallne-23 of the topoisomerase 11β-1 protein sequence. The topoisomerase 11β-1 and β-2 mRNAs were both widely expressed in human cell lines and tissues. Topoisomerase IIβ-2 mRNA was expressed at a lower level than that of the β-1 form, but the relative expression of the two forms varied in different cell types. Analysis of genomlc DNA clones revealed that the two forms of topoisomerase IIβ mRNA arose via differential splicing. These data indicate that in addition to the closely related topoisomerase Ha and β isozymes, there are two forms of topoisomerase IIβ mRNA widely expressed in human cells.
