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Ribosomal DNA (rDNA) encodes the ribosomal RNA genes and represents an intrinsically unstable genomic region. However, the underlying mechanisms and implications for genome integrity remain elusive. Here, we use Bloom syndrome (BS), a rare genetic disease characterized by DNA repair defects and hyper-unstable rDNA, as a model to investigate the mechanisms leading to rDNA instability. We find that in Bloom helicase (BLM) proficient cells, the homologous recombination (HR) pathway in rDNA resembles that in nuclear chromatin; it is initiated by resection, replication protein A (RPA) loading and BRCA2-dependent RAD51 filament formation. However, BLM deficiency compromises RPA-loading and BRCA1/2 recruitment to rDNA, but not RAD51 accumulation. RAD51 accumulates at rDNA despite depletion of long-range resection nucleases and rDNA damage results in micronuclei when BLM is absent. In summary, our findings indicate that rDNA is permissive to RAD51 accumulation in the absence of BLM, leading to micronucleation and potentially global genomic instability., (© 2024. The Author(s).)

Generation of O 6 -methylguanine (O 6 -meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O 6 -meG-induced apoptosis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ryosuke Fujikane reports financial support was provided by JSPS KAKENHI. Masumi Hidaka reports financial support was provided by JSPS KAKENHI. Masumi Hidaka reports financial support was provided by Promotion and Mutual Aid Corporation for Private Schools of Japan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers., Competing Interests: Declaration of interests R.A.G. is a co-founder and scientific advisory board member of RADD Pharmaceuticals and a scientific advisory board member for Dong-A ST Co. Neither engagement directly relates to the substance of this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bloom syndrome helicase (BLM) is a RecQ-family helicase implicated in a variety of cellular processes, including DNA replication, DNA repair, and telomere maintenance. Mutations in human BLM cause Bloom syndrome (BS), an autosomal recessive disorder that leads to myriad negative health impacts including a predisposition to cancer. BS-causing mutations in BLM often negatively impact BLM ATPase and helicase activity. While BLM mutations that cause BS have been well characterized both in vitro and in vivo, there are other less studied BLM mutations that exist in the human population that do not lead to BS. Two of these non-BS mutations, encoding BLM P868L and BLM G1120R, when homozygous, increase sister chromatid exchanges in human cells. To characterize these naturally occurring BLM mutant proteins in vitro, we purified the BLM catalytic core (BLMcore, residues 636-1298) with either the P868L or G1120R substitution. We also purified a BLMcore K869A K870A mutant protein, which alters a lysine-rich loop proximal to the P868 residue. We found that BLMcore P868L and G1120R proteins were both able to hydrolyze ATP, bind diverse DNA substrates, and unwind G-quadruplex and duplex DNA structures. Molecular dynamics simulations suggest that the P868L substitution weakens the DNA interaction with the winged-helix domain of BLM and alters the orientation of one lobe of the ATPase domain. Because BLMcore P868L and G1120R retain helicase function in vitro, it is likely that the increased genome instability is caused by specific impacts of the mutant proteins in vivo. Interestingly, we found that BLMcore K869A K870A has diminished ATPase activity, weakened binding to duplex DNA structures, and less robust helicase activity compared to wild-type BLMcore. Thus, the lysine-rich loop may have an important role in ATPase activity and specific binding and DNA unwinding functions in BLM., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Cueny et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Homologous recombination (HR) is a key DNA damage repair pathway that is tightly adjusted to the state of a cell. A central regulator of homologous recombination is the conserved helicase-containing Bloom syndrome complex, renowned for its crucial role in maintaining genome integrity. Here, we show that in Arabidopsis thaliana, Bloom complex activity is controlled by selective autophagy. We find that the recently identified DNA damage regulator KNO1 facilitates K63-linked ubiquitination of RMI1, a structural component of the complex, thereby triggering RMI1 autophagic degradation and resulting in increased homologous recombination. Conversely, reduced autophagic activity makes plants hypersensitive to DNA damage. KNO1 itself is also controlled at the level of proteolysis, in this case mediated by the ubiquitin-proteasome system, becoming stabilized upon DNA damage via two redundantly acting deubiquitinases, UBP12 and UBP13. These findings uncover a regulatory cascade of selective and interconnected protein degradation steps resulting in a fine-tuned HR response upon DNA damage., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Prostate cancer (PCa) is a common cancer and a major cause of cancer-related death worldwide in men, necessitating novel targets for cancer therapy. High expression of Bloom's syndrome protein (BLM) helicase is associated with the occurrence and development of PCa. Therefore, the identification and development of new BLM inhibitors may be a new direction for the treatment of PCa. Here, we identified a novel inhibitor by molecular docking and put it to systematic evaluation via various experiments, AO/854, which acted as a competitive inhibitor that blocked the BLM-DNA interaction. Cellular evaluation indicated that AO/854-suppressed tumor growth and metastasis in PC3 cells by enhancing DNA damage, phosphorylating Chk1/Chk2, and altering the p53 signaling pathway. Collectively, the study highlights the potential of BLM as a therapeutic target in PCa and reveals a distinct mechanism by which AO/854 competitively inhibits the function of BLM.

Bloom syndrome (BS) is an autosomal recessive disease clinically characterized by primary microcephaly, growth deficiency, immunodeficiency and predisposition to cancer. It is mainly caused by biallelic loss-of-function mutations in the BLM gene, which encodes the BLM helicase, acting in DNA replication and repair processes. Here, we describe the gene expression profiles of three BS fibroblast cell lines harboring causative, biallelic truncating mutations obtained by single-cell (sc) transcriptome analysis. We compared the scRNA transcription profiles from three BS patient cell lines to two age-matched wild-type controls and observed specific deregulation of gene sets related to the molecular processes characteristically affected in BS, such as mitosis, chromosome segregation, cell cycle regulation and genomic instability. We also found specific upregulation of genes of the Fanconi anemia pathway, in particular FANCM, FANCD2 and FANCI, which encode known interaction partners of BLM. The significant deregulation of genes associated with inherited forms of primary microcephaly observed in our study might explain in part the molecular pathogenesis of microcephaly in BS, one of the main clinical characteristics in patients. Finally, our data provide first evidence of a novel link between BLM dysfunction and transcriptional changes in condensin complex I and II genes. Overall, our study provides novel insights into gene expression profiles in BS on an sc level, linking specific genes and pathways to BLM dysfunction., (© The Author(s) 2022. Published by Oxford University Press.)

Bloom's syndrome is caused by inactivation of the BLM helicase, which functions with TOP3A and RMI1-2 (BTR complex) to dissolve recombination intermediates and avoid somatic crossing-over. We show here that crossover avoidance by BTR further requires the activity of cyclin-dependent kinase-1 (CDK1), Polo-like kinase-1 (PLK1), and the DDR mediator protein TOPBP1, which act in the same pathway. Mechanistically, CDK1 phosphorylates BLM and TOPBP1 and promotes the interaction of both proteins with PLK1. This is amplified by the ability of TOPBP1 to facilitate phosphorylation of BLM at sites that stimulate both BLM-PLK1 and BLM-TOPBP1 binding, creating a positive feedback loop that drives rapid BLM phosphorylation at the G 2 -M transition. In vitro, BLM phosphorylation by CDK/PLK1/TOPBP1 stimulates the dissolution of topologically linked DNA intermediates by BLM-TOP3A. Thus, we propose that the CDK1-TOPBP1-PLK1 axis enhances BTR-mediated dissolution of recombination intermediates late in the cell cycle to suppress crossover recombination and curtail genomic instability.

TOP3A promotes processing of double Holliday junction dissolution and also plays an important role in decatenation and segregation of human mtDNA. Recently, TOP3A mutations have been reported to cause Bloom syndrome-like disorder. However, whether the two function play equal roles in the disease pathogenesis is unclear. We retrospectively studied the disease progression of two siblings with Bloom-like syndrome caused by two novel mutations of TOP3A, p.Q788* and p.D479G. Beside the common clinical manifestations, our patients exhibited liver lipid storage with hepatomegaly. In cellular and molecular biological studies, TOP3A deficiency moderately increased sister chromatid exchanges and decreased cell proliferation compared with BLM or RMI2 deficiency. These changes were rescued by ectopic expression of either of the wildtype TOP3A or TOP3A-D479G. In contrast, reduced mitochondrial ATP generation and oxygen consumption rates observed in TOP3A defective cells were rescued by over-expression of the wildtype TOP3A, but not TOP3A-D479G. Considering the different impact of the TOP3A-D479G mutation on the genome stability and mitochondrial metabolism, we propose that the impaired mitochondrial metabolism plays an important role in the pathogenesis of TOP3A-deficient Bloom-like disease., (Copyright © 2021. Published by Elsevier B.V.)

Werner Syndrome (WS) and Bloom Syndrome (BS) are disorders of DNA damage repair caused by biallelic disruption of the WRN or BLM DNA helicases respectively. Both are commonly associated with insulin resistant diabetes, usually accompanied by dyslipidemia and fatty liver, as seen in lipodystrophies. In keeping with this, progressive reduction of subcutaneous adipose tissue is commonly observed. To interrogate the underlying cause of adipose tissue dysfunction in these syndromes, CRISPR/Cas9 genome editing was used to generate human pluripotent stem cell (hPSC) lacking either functional WRN or BLM helicase. No deleterious effects were observed in WRN -/- or BLM -/- embryonic stem cells, however upon their differentiation into adipocyte precursors (AP), premature senescence emerged, impairing later stages of adipogenesis. The resulting adipocytes were also found to be senescent, with increased levels of senescent markers and senescence-associated secretory phenotype (SASP) components. SASP components initiate and reinforce senescence in adjacent cells, which is likely to create a positive feedback loop of cellular senescence within the adipocyte precursor compartment, as demonstrated in normal ageing. Such a scenario could progressively attenuate adipose mass and function, giving rise to "lipodystrophy-like" insulin resistance. Further assessment of pharmacological senolytic strategies are warranted to mitigate this component of Werner and Bloom syndromes.

Mutations in BLM helicase predispose Bloom syndrome (BS) patients to a wide spectrum of cancers. We demonstrate that MIB1-ubiquitylated BLM in G1 phase functions as an adaptor protein by enhancing the binding of transcription factor c-Jun and its E3 ligase, Fbw7α. BLM enhances the K48/K63-linked ubiquitylation on c-Jun, thereby enhancing the rate of its subsequent degradation. Functionally defective Fbw7α mutants prevalent in multiple human cancers are reactivated by BLM. However, BS patient-derived BLM mutants cannot potentiate Fbw7α-dependent c-Jun degradation. The decrease in the levels of c-Jun in cells expressing BLM prevents effective c-Jun binding to 2,584 gene promoters. This causes decreases in the transcript and protein levels of c-Jun targets in BLM-expressing cells, resulting in attenuated c-Jun-dependent effects during neoplastic transformation. Thus, BLM carries out its function as a tumor suppressor by enhancing c-Jun turnover and thereby preventing its activity as a proto-oncogene., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sgs1, the orthologue of human Bloom's syndrome helicase BLM, is a yeast DNA helicase functioning in DNA replication and repair. We show that SGS1 loss increases R-loop accumulation and sensitizes cells to transcription-replication collisions. Yeast lacking SGS1 accumulate R-loops and γ-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1 Δ reveals copy number changes flanked by repetitive regions with high R-loop-forming potential. Analysis of BLM in Bloom's syndrome fibroblasts or by depletion of BLM from human cancer cells confirms a role for Sgs1/BLM in suppressing R-loop-associated genome instability across species. In support of a potential direct effect, BLM is found physically proximal to DNA:RNA hybrids in human cells, and can efficiently unwind R-loops in vitro. Together, our data describe a conserved role for Sgs1/BLM in R-loop suppression and support an increasingly broad view of DNA repair and replication fork stabilizing proteins as modulators of R-loop-mediated genome instability., (© 2017 Chang et al.)

Self-catalyzed DNA depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5'G residue of the 5'GA/TGG loop and of the first 5'A residue of the 5'GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000-4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenicity at such sites in many human genes is associated with loss of function of key proteins responsible for diverse diseases.

Rare pleiotropic genetic disorders, Ataxia-telangiectasia (A-T), Bloom syndrome (BS) and Nijmegen breakage syndrome (NBS) are characterised by immunodeficiency, extreme radiosensitivity, higher cancer susceptibility, premature aging, neurodegeneration and insulin resistance. Some of these functional abnormalities can be explained by aberrant DNA damage response and chromosomal instability. It has been suggested that one possible common denominator of these conditions could be chronic oxidative stress caused by endogenous ROS overproduction and impairment of mitochondrial homeostasis. Recent studies indicate new, alternative sources of oxidative stress in A-T, BS and NBS cells, including NADPH oxidase 4 (NOX4), oxidised low-density lipoprotein (ox-LDL) or Poly (ADP-ribose) polymerases (PARP). Mitochondrial abnormalities such as changes in the ultrastructure and function of mitochondria, excess mROS production as well as mitochondrial damage have also been reported in A-T, BS and NBS cells. A-T, BS and NBS cells are inextricably linked to high levels of reactive oxygen species (ROS), and thereby, chronic oxidative stress may be a major phenotypic hallmark in these diseases. Due to the presence of mitochondrial disturbances, A-T, BS and NBS may be considered mitochondrial diseases. Excess activity of antioxidant enzymes and an insufficient amount of low molecular weight antioxidants indicate new pharmacological strategies for patients suffering from the aforementioned diseases. However, at the current stage of research we are unable to ascertain if antioxidants and free radical scavengers can improve the condition or prolong the survival time of A-T, BS and NBS patients. Therefore, it is necessary to conduct experimental studies in a human model., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Bloom syndrome is a rare and recessive disorder characterized by loss-of-function mutations of the BLM gene, which encodes a RecQ 3'-5' DNA helicase. Despite its putative tumor suppressor function, the contribution of BLM to human sporadic colorectal cancer (CRC) remains poorly understood., Methods: The transcriptional regulation mechanism underlying BLM and related DNA damage response regulation in independent CRC subsets and a panel of derived cell lines was investigated by bioinformatics analysis, the transcriptomic profile, a CpG island promoter methylation assay, Western blot, and an immunolocalization assay., Results: In silico analysis of gene expression data sets revealed that BLM is overexpressed in poorly differentiated CRC and exhibits a close connection with shorter relapse-free survival even after adjustment for prognostic factors and pathways that respond to DNA damage response through ataxia telangiectasia mutated (ATM) signaling. Functional characterization demonstrated that CpG island promoter hypomethylation increases BLM expression and associates with cytoplasmic BLM mislocalization and increased DNA damage response both in clinical CRC samples and in derived cancer cell lines. The DNA-damaging agent S-adenosylmethionine suppresses BLM expression, leading to the inhibition of cell growth following accumulation of DNA damage. In tumor specimens, cytoplasmic accumulation of BLM correlates with DNA damage and γH2AX and phosphorylated ATM foci and predicts long-term progression-free survival in metastatic patients treated with irinotecan., Conclusions: Taken together, the findings of this study provide the first evidence that cancer-linked DNA hypomethylation and cytosolic BLM mislocalization might reflect compromised levels of DNA-repair activity and enhanced hypersensitivity to DNA-damaging agents in CRC patients.

Sister chromatid exchanges (SCEs) are considered sensitive indicators of genome instability. Detection of SCEs typically requires cells to incorporate bromodeoxyuridine (BrdU) during two rounds of DNA synthesis. Previous studies have suggested that SCEs are induced by DNA replication over BrdU-substituted DNA and that BrdU incorporation alone could be responsible for the high number of SCE events observed in cells from patients with Bloom syndrome (BS), a rare genetic disorder characterized by marked genome instability and high SCE frequency. Here we show using Strand-seq, a single cell DNA template strand sequencing technique, that the presence of variable BrdU concentrations in the cell culture medium and in DNA template strands has no effect on SCE frequency in either normal or BS cells. We conclude that BrdU does not induce SCEs and that SCEs detected in either normal or BS cells reflect DNA repair events that occur spontaneously., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chromosome instability usually leads to tumorigenesis. Bloom syndrome (BS) is a genetic disease associated with chromosome instability. The BS gene product, BLM, has been reported to function in the spindle assembly checkpoint (SAC) to prevent chromosome instability. BTR complex, composed of BLM, topoisomerase IIIα (Topo IIIα), RMI1 (RecQ-mediated genome instability protein 1, BLAP75) and RMI2 (RecQ-mediated genome instability protein 2, BLAP18), is crucial for maintaining genome stability. Recent work has demonstrated that RMI2 also plays critical role in SAC. However, little is know about RMI1 regulation during the cell cycle. Here we present that RMI1 protein level does not change through G1, S and G2 phases, but significantly increases in M phase. Moreover, phosphorylation of RMI1 occurs in mitosis. Upon microtubule-disturbing agent, RMI1 is phosphorylated primarily at the sites of Serine 284 and Serine 292, which does not interfere with the formation of BTR complex. Additionally, this phosphorylation is partially reversed by roscovitine treatment, implying cycling-dependent kinase 1 (CDK1) might be one of the upstream kinases.

The Bloom syndrome (BS) is an autosomal recessive disorder associated with dwarfism, immunodeficiency, reduced fertility and cancer risk. BS cells show genomic instability, particularly an hyper exchange between the sister chromatids due to a defective processing of the DNA replication intermediates. It is caused by mutations in the BLM gene which encodes a member of the RecQ family of DExH box DNA helicases. In this study, we reported cytogenetic, BLM linkage and mutational analyses for two affected Tunisian families. The Cytogenetic parameters were performed by chromosomal aberration (CA) and sister chromatid exchange (SCE) assays and results showed a significant increase in mean frequency of CA and SCE in BS cells. BLM linkage performed by microsatellite genotyping revealed homozygous haplotypes for the BS patients, evidence of linkage to BLM gene. Mutational analysis by direct DNA sequencing revealed a novel frameshift mutation (c.1980-1982delAA) in exon 8 of BLM gene, resulting in a truncated protein (p.Lys662fsX5). The truncated protein could explain genomic instability and its related symptoms in the BS patients. The screening of this mutation is useful for BS diagnosis confirmation in Tunisian families., (© 2013 European Academy of Dermatology and Venereology.)

Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.

The Bloom syndrome gene product, BLM, is a member of the highly conserved RecQ family. An emerging concept is the BLM helicase collaborates with the homologous recombination (HR) machinery to help avoid undesirable HR events and to achieve a high degree of fidelity during the HR reaction. However, exactly how such coordination occurs in vivo is poorly understood. Here, we identified a protein termed SPIDR (scaffolding protein involved in DNA repair) as the link between BLM and the HR machinery. SPIDR independently interacts with BLM and RAD51 and promotes the formation of a BLM/RAD51-containing complex of biological importance. Consistent with its role as a scaffolding protein for the assembly of BLM and RAD51 foci, cells depleted of SPIDR show increased rate of sister chromatid exchange and defects in HR. Moreover, SPIDR depletion leads to genome instability and causes hypersensitivity to DNA damaging agents. We propose that, through providing a scaffold for the cooperation of BLM and RAD51 in a multifunctional DNA-processing complex, SPIDR not only regulates the efficiency of HR, but also dictates the specific HR pathway.

Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Defects in the human BLM gene cause Bloom syndrome, notable for early development of tumors in a broad variety of tissues. On the basis of sequence similarity, BLM has been identified as one of the five human homologs of RecQ from Escherichia coli. Nevertheless, biochemical characterization of the BLM protein indicates far greater functional similarity to the E. coli RecG protein and there is no known RecG homolog in human cells. To explore the possibility that the shared biochemistries of BLM and RecG may represent an example of convergent evolution of cellular function where in humans BLM has evolved to fulfill the genomic stabilization role of RecG, we determined whether expression of RecG in human BLM-deficient cells could suppress established functional cellular Bloom syndrome phenotypes. We found that RecG can indeed largely suppress both the definitive elevated sister chromatid exchange phenotype and the more recently demonstrated gene cluster instability phenotype of BLM-deficient cells. In contrast, expression of RecG has no impact on either of these phenotypes in human cells with functional BLM protein. These results suggest that the combination of biochemical activities shared by RecG and BLM fill the same evolutionary niche in preserving genomic integrity without requiring exactly identical molecular mechanisms.

The RMI subcomplex (RMI1/RMI2) functions with the BLM helicase and topoisomerase IIIα in a complex called the "dissolvasome," which separates double-Holliday junction DNA structures that can arise during DNA repair. This activity suppresses potentially harmful sister chromatid exchange (SCE) events in wild-type cells but not in cells derived from Bloom syndrome patients with inactivating BLM mutations. The RMI subcomplex also associates with FANCM, a component of the Fanconi anemia (FA) core complex that is important for repair of stalled DNA replication forks. The RMI/FANCM interface appears to help coordinate dissolvasome and FA core complex activities, but its precise role remains poorly understood. Here, we define the structure of the RMI/FANCM interface and investigate its roles in coordinating cellular DNA-repair activities. The X-ray crystal structure of the RMI core complex bound to a well-conserved peptide from FANCM shows that FANCM binds to both RMI proteins through a hydrophobic "knobs-into-holes" packing arrangement. The RMI/FANCM interface is shown to be critical for interaction between the components of the dissolvasome and the FA core complex. FANCM variants that substitute alanine for key interface residues strongly destabilize the complex in solution and lead to increased SCE levels in cells that are similar to those observed in blm- or fancm-deficient cells. This study provides a molecular view of the RMI/FANCM complex and highlights a key interface utilized in coordinating the activities of two critical eukaryotic DNA-damage repair machines.

Bloom syndrome (BS) is a rare autosomal genetic disorder characterized by lupus-like erythematous telangi-ectasias of the face, sun sensitivity, infertility, stunted growth, upper respiratory infection, and gastrointestinal infections commonly associated with decreased immuno-globulin levels. The syndrome is associated with immuno-deficiency of a generalized type, ranging from mild and essentially asympto-matic to severe. Chromosomal abnormalities are hallmarks of the disorder, and high frequencies of sister chromatid exchanges and quadriradial configurations in lymphocytes and fibroblasts are diagnostic features. BS is caused by mutations in BLM, a member of the RecQ helicase family. We determined whether BLM deficiency has any effects on cell growth and death in BLM-deficient cells and mice. BLM-deficient EB-virus-transformed cell lines from BS patients and embryonic fibroblasts from BLM-/- mice showed slower growth than wild-type cells. BLM-deficient cells showed abnormal p53 protein expression after irradiation. In BLM-/- mice, small body size, reduced number of fetal liver cells and increased cell death were observed. BLM deficiency causes the up-regulation of p53, double-strand break and apoptosis, which are likely observed in irradiated control cells. Slow cell growth and increased cell death may be one of the causes of the small body size associated with BS patients.

The maintenance of genome stability is critical for the suppression of cancer and premature ageing. The maintenance of the human genome requires hundreds of proteins involved in DNA repair, DNA replication, chromosome segregation and cell cycle checkpoint responses. A number of genetic disorders exist in man where a breakdown in genome maintenance is associated with cancer predisposition. Amongst these are Bloom's syndrome (BS) and Fanconi anaemia (FA). The BS and FA gene products co-operate in the repair of damaged DNA. In this review, we focus on interactions between BS and FA proteins that specifically occur during chromosome segregation in mitosis. The BS protein, BLM, was shown recently to define a novel class of anaphase DNA bridge structures that, in some cases, also contain FA proteins. We will discuss the possible source of these bridges and the role that FA proteins and BLM might play in their removal.

Genetic instability due to increased DNA damage and altered DNA repair is of central significance in the initiation and progression of inherited and sporadic human leukemias. Although very rare, some inherited DNA repair insufficiency syndromes (e.g., Fanconi anemia, Bloom's syndrome) have added substantially to our understanding of crucial mechanisms of leukemogenesis in recent years. Conversely, sporadic leukemias account for the main proportion of leukemias and here DNA damaging reactive oxygen species (ROS) play a central role. Although the exact mechanisms of increased ROS production remain largely unknown and no single pathway has been detected thus far, some oncogenic proteins (e.g., the activated tyrosine kinases BCR-ABL1 and FLT3-ITD) seem to play a key role in driving genetic instability by increased ROS generation which influences the disease course (e.g., blast crisis in chronic myeloid leukemia or relapse in FLT3-ITD positive acute myeloid leukemia). Of course other mechanisms, which promote genetic instability in leukemia also exist. A newly emerging mechanism is the genome-wide alteration of epigenetic marks (e.g., hypomethylation of histone H3K79), which promotes chromosomal instability. Taken together genetic instability plays a critical role both in inherited and sporadic leukemias and emerges as a common theme in both inherited and sporadic leukemias. Beyond its theoretical impact, the analysis of genetic instability may lead the way to the development of innovative therapy strategies., (© 2010 Wiley-Liss, Inc.)

Mutations in BLM, a RecQ-like helicase, are linked to the autosomal recessive cancer-prone disorder Bloom's syndrome. BLM associates with topoisomerase (Topo) IIIα, RMI1, and RMI2 to form the BLM complex that is essential for genome stability. The RMI1-RMI2 heterodimer stimulates the dissolution of double Holliday junction into non-crossover recombinants mediated by BLM-Topo IIIα and is essential for stabilizing the BLM complex. However, the molecular basis of these functions of RMI1 and RMI2 remains unclear. Here we report the crystal structures of multiple domains of RMI1-RMI2, providing direct confirmation of the existence of three oligonucleotide/oligosaccharide binding (OB)-folds in RMI1-RMI2. Our structural and biochemical analyses revealed an unexpected insertion motif in RMI1N-OB, which is important for stimulating the dHJ dissolution. We also revealed the structural basis of the interaction between RMI1C-OB and RMI2-OB and demonstrated the functional importance of the RMI1-RMI2 interaction in genome stability maintenance., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

BLM, the protein product of the gene mutated in Bloom syndrome, is one of five human RecQ helicases. It functions to separate double Holliday junction DNA without genetic exchange as a component of the "dissolvasome," which also includes topoisomerase IIIα and the RMI (RecQ-mediated genome instability) subcomplex (RMI1 and RMI2). We describe the crystal structure of the RMI core complex, comprising RMI2 and the C-terminal OB domain of RMI1. The overall RMI core structure strongly resembles two-thirds of the trimerization core of the eukaryotic single-stranded DNA-binding protein, Replication Protein A. Immunoprecipitation experiments with RMI2 variants confirm key interactions that stabilize the RMI core interface. Disruption of this interface leads to a dramatic increase in cellular sister chromatid exchange events similar to that seen in BLM-deficient cells. The RMI core interface is therefore crucial for BLM dissolvasome assembly and may have additional cellular roles as a docking hub for other proteins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their Saccharomyces cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1Delta mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA.

Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by a strong predisposition to a wide range of cancers commonly affecting the general population. Understanding the functioning mechanism of the BLM protein may provide the opportunity to develop new effective therapy strategies. In this work, we studied the DNA unwinding kinetic mechanism of the helicase core of the BLM protein using various stopped-flow assays. We show that the helicase core of BLM unwinds duplex DNA as monomers even under conditions strongly favoring oligomerization. An unwinding rate of approximately 20 steps per second and a step size of 1 bp have been determined. We have observed that the helicase has a very low processivity. From dissociation and inhibition experiments, we have found that during its ATP hydrolysis cycle in DNA unwinding the helicase tends to dissociate from the DNA substrate in the ADP state. The experimental results imply that the BLM helicase core may unwind duplex DNA in an inchworm manner.

Fanconi Anemia (FA) and Bloom's Syndrome (BS) are genetic disorders characterized by overlapping phenotypes, including aberrant DNA repair and cancer predisposition. Here, we show that the FANCM gene product, FANCM protein, links FA and BS by acting as a protein anchor and bridge that targets key components of the FA and BS pathways to stalled replication forks, thus linking multiple components that are necessary for efficient DNA repair. Two highly conserved protein:protein interaction motifs in FANCM, designated MM1 and MM2, were identified. MM1 interacts with the FA core complex by binding to FANCF, whereas MM2 interacts with RM1 and topoisomerase IIIalpha, components of the BS complex. The MM1 and MM2 motifs were independently required to activate the FA and BS pathways. Moreover, a common phenotype of BS and FA cells-an elevated frequency of sister chromatid exchanges-was due to a loss of interaction of the two complexes through FANCM., (2009 Elsevier Inc.)

In this issue of Molecular Cell, Deans and West (2009) reveal the molecular basis of the phenotypic similarities between Fanconi Anemia (FA) and Bloom's Syndrome, identifying FANCM as the anchor for both FA and Bloom's complexes at the site of the DNA interstrand crosslink., (2009 Elsevier Inc.)

Loss-of-function of caretaker genes characterizes a group of cancer predisposition diseases that feature cellular hypersensitivity to DNA damage and chromosome fragility; this group includes Fanconi anaemia and Bloom syndrome. The products of the 13 FANC genes (mutated in Fanconi anaemia), which constitute the 'FANC' pathway, and BLM (the RecQ helicase mutated in Bloom syndrome) are thought to collaborate during the S phase of the cell cycle, preventing chromosome instability. Recently, BLM has been implicated in the completion of sister chromatid separation during mitosis, a complex process in which precise regulation and execution is crucial to preserve genomic stability. Here we show for the first time a role for the FANC pathway in chromosome segregation during mitotic cell division. FANCD2, a key component of the pathway, localizes to discrete spots on mitotic chromosomes. FANCD2 chromosomal localization is responsive to replicative stress and specifically targets aphidicolin (APH)-induced chromatid gaps and breaks. Our data indicate that the FANC pathway is involved in rescuing abnormal anaphase and telophase (ana-telophase) cells, limiting aneuploidy and reducing chromosome instability in daughter cells. We further address a cooperative role for the FANC pathway and BLM in preventing micronucleation, through FANC-dependent targeting of BLM to non-centromeric abnormal structures induced by replicative stress. We reveal new crosstalk between FANC and BLM proteins, extending their interaction beyond the S-phase rescue of damaged DNA to the safeguarding of chromosome stability during mitosis.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by genomic instability and cancer predisposition. The underlying genetic defect is mutation of the BLM gene, producing deficiency in the RecQ helicase BLM (Bloom's syndrome protein). The present article begins by introducing BLM and its binding partners before reviewing its known biochemical activities and its potential roles both as a pro-recombinase and as a suppressor of homologous recombination. Finally, the evidence for an emerging role in mitotic chromosome segregation is examined.

The use of co-immunoprecipitation (co-IP) to purify multi-protein complexes has contributed greatly to our understanding of the DNA damage response network associated with Fanconi anemia (FA), Bloom syndrome (BS) and breast cancer. Four new FA genes and two new protein partners for the Bloom syndrome gene product have been identified by co-IP. Here, we discuss our experience in using co-IP and other techniques to isolate and characterize new FA and BS-related proteins.

Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.

BLM, the helicase mutated in Bloom syndrome, associates with topoisomerase 3alpha, RMI1 (RecQ-mediated genome instability), and RPA, to form a complex essential for the maintenance of genome stability. Here we report a novel component of the BLM complex, RMI2, which interacts with RMI1 through two oligonucleotide-binding (OB)-fold domains similar to those in RPA. The resulting complex, named RMI, differs from RPA in that it lacks obvious DNA-binding activity. Nevertheless, RMI stimulates the dissolution of a homologous recombination intermediate in vitro and is essential for the stability, localization, and function of the BLM complex in vivo. Notably, inactivation of RMI2 in chicken DT40 cells results in an increased level of sister chromatid exchange (SCE)--the hallmark feature of Bloom syndrome cells. Epistasis analysis revealed that RMI2 and BLM suppress SCE within the same pathway. A point mutation in the OB domain of RMI2 disrupts the association between BLM and the rest of the complex, and abrogates the ability of RMI2 to suppress elevated SCE. Our data suggest that multi-OB-fold complexes mediate two modes of BLM action: via RPA-mediated protein-DNA interaction, and via RMI-mediated protein-protein interactions.

Bloom Syndrome is an autosomal recessive cancer-prone disorder caused by mutations in the BLM gene. BLM encodes a DNA helicase of the RECQ family, and associates with Topo IIIalpha and BLAP75/RMI1 (BLAP for BLM-associated polypeptide/RecQ-mediated genome instability) to form the BTB (BLM-Topo IIIalpha-BLAP75/RMI1) complex. This complex can resolve the double Holliday junction (dHJ), a DNA intermediate generated during homologous recombination, to yield noncrossover recombinants exclusively. This attribute of the BTB complex likely serves to prevent chromosomal aberrations and rearrangements. Here we report the isolation and characterization of a novel member of the BTB complex termed BLAP18/RMI2. BLAP18/RMI2 contains a putative OB-fold domain, and several lines of evidence suggest that it is essential for BTB complex function. First, the majority of BLAP18/RMI2 exists in complex with Topo IIIalpha and BLAP75/RMI1. Second, depletion of BLAP18/RMI2 results in the destabilization of the BTB complex. Third, BLAP18/RMI2-depleted cells show spontaneous chromosomal breaks and are sensitive to methyl methanesulfonate treatment. Fourth, BLAP18/RMI2 is required to target BLM to chromatin and for the assembly of BLM foci upon hydroxyurea treatment. Finally, BLAP18/RMI2 stimulates the dHJ resolution capability of the BTB complex. Together, these results establish BLAP18/RMI2 as an essential member of the BTB dHJ dissolvasome that is required for the maintenance of a stable genome.

A key cellular response to DNA double-strand breaks (DSBs) is 5'-to-3' DSB resection by nucleases to generate regions of ssDNA that then trigger cell cycle checkpoint signaling and DSB repair by homologous recombination (HR). Here, we reveal that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae RecQ-family helicase, Sgs1, causes pronounced hypersensitivity to DSB-inducing agents. Moreover, we establish that this reflects severely compromised DSB resection, deficient DNA damage signaling, and strongly impaired HR-mediated repair. Furthermore, we show that the mammalian Sgs1 ortholog, BLM--whose deficiency causes cancer predisposition and infertility in people--also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents. Collectively, these data establish evolutionarily conserved roles for the BLM and Sgs1 helicases in DSB processing, signaling, and repair.

Replication checkpoint protein Rad17 senses DNA lesions during DNA replication and halts progression of replication fork. The cells derived from Bloom syndrome individuals show some defects in DNA replication. In order to investigate the functional relationship between the replication checkpoint protein Rad17 and BLM, which is the product of the causative gene of Bloom syndrome, we generated BLM/RAD17 double knockout (blm/rad17) cells using chicken DT40 cells. The blm/rad17 cells showed exaggerated growth defects as determined by analysis of their growth curves and plating efficiency compared to those of either of the single gene mutants. These defects seem to be due to an increase in DNA lesions that cause spontaneous cell death, suggesting that Rad17 and BLM execute different functions in the progression of replication forks. We also demonstrate that targeting integration was dramatically compromised by a lack of Rad17. In addition, the elevated frequency of sister chromatid exchange (SCE) due to homologous recombination in BLM knockout (blm) cells was greatly reduced by disruption of the RAD17 gene. Thus, in addition to its role in the replication checkpoint, Rad17 appears to play a role in homologous recombination.

Bloom syndrome is a genetic disorder associated with chromosomal instability and a predisposition to tumors that is caused by germline mutations of the BLM gene, a RecQ helicase. Benign adrenocortical tumors display a degree of chromosomal instability that is more significant than benign tumors of other tissues. Cortisol-producing hyperplasias, such as primary pigmented nodular adrenocortical disease (PPNAD), which has been associated with protein kinase A (PKA) abnormalities and/or PRKAR1A mutations, also show genomic instability. Another RecQ helicase, WRN, directly interacts with the PRKAR1B subunit of PKA. In this study, we have investigated the PRKAR1A expression in primary human Bloom syndrome cell lines with known BLM mutations and examined the BLM gene expression in PPNAD and other adrenal tumor tissues. PRKAR1A and other protein kinase A (PKA) subunits were expressed in Bloom syndrome cells and their level of expression differed by subunit and cell type. Overall, fibroblasts exhibited a significant decrease in protein expression of all PKA subunits except for PRKAR1A, a pattern that has been associated with neoplastic transformation in several cell types. The BLM protein was upregulated in PPNAD and other hyperplasias, compared to samples from normal adrenals and normal cortex, as well as samples from cortisol- and aldosterone-producing adenomas (in which BLM was largely absent). These data reveal an inverse relationship between BLM and PRKAR1A: BLM deficiency is associated with a relative excess of PRKAR1A in fibroblasts compared to other PKA subunits; and PRKAR1A deficiency is associated with increased BLM protein in adrenal hyperplasias.

A comparative evaluation is reported of pro-oxidant states in 82 patients with ataxia telangectasia (AT), Bloom syndrome (BS), Down syndrome (DS), Fanconi anemia (FA), Werner syndrome (WS), and xeroderma pigmentosum (XP) vs 98 control donors. These disorders display cancer proneness, and/or early aging, and/or other clinical features. The measured analytes were: (a) leukocyte and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG), (b) blood glutathione (GSSG and GSH), (c) plasma glyoxal (Glx) and methylglyoxal (MGlx), and (d) some plasma antioxidants [uric acid (UA) and ascorbic acid (AA)]. Leukocyte 8-OHdG levels ranked as follows: WS>BS approximately FA approximately XP>DS approximately AT approximately controls. Urinary 8-OHdG levels were significantly increased in a total of 22 patients with BS, FA, or XP vs 47 controls. The GSSG:GSH ratio was significantly increased in patients with WS and in young (< or =15 years) patients with DS or with FA and decreased in older patients with DS or FA and in AT, BS, and XP patients. The plasma levels of Glx and/or MGlx were significantly increased in patients with WS, FA, and DS. The UA and AA levels were significantly increased in WS and DS patients, but not in AT, FA, BS, nor XP patients. Rationale for chemoprevention trials is discussed.

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 ataxia telangiectasia mutated and Rad3-related (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 alpha 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.

Bloom's syndrome (BS) is an autosomal recessive disorder characterized by a strong cancer predisposition. The defining feature of BS is extreme genome instability. The gene mutated in Bloom's syndrome, BLM, encodes a DNA helicase (BLM) of the RecQ family. BLM plays a role in homologous recombination; however, its exact function remains controversial. Mutations in the BLM cause hyperrecombination between sister chromatids and homologous chromosomes, indicating an anti-recombination role. Conversely, other data show that BLM is required for recombination. It was previously shown that in vitro BLM helicase promotes disruption of recombination intermediates, regression of stalled replication forks, and dissolution of double Holliday junctions. Here, we demonstrate two novel activities of BLM: disruption of the Rad51-ssDNA (single-stranded DNA) filament, an active species that promotes homologous recombination, and stimulation of DNA repair synthesis. Using in vitro reconstitution reactions, we analyzed how different biochemical activities of BLM contribute to its functions in homologous recombination.

BLM, the protein mutated in Bloom's syndrome, possesses a helicase activity that can dissociate DNA structures, including the Holliday junction, expected to arise during homologous recombination. BLM is stably associated with topoisomerase IIIalpha (Topo IIIalpha) and the BLAP75 protein. The BLM-Topo IIIalpha-BLAP75 (BTB) complex can efficiently resolve a DNA substrate that harbors two Holliday junctions (the double Holliday junction) in a non-crossover manner. Here we show that the Holliday junction unwinding activity of BLM is greatly enhanced as a result of its association with Topo IIIalpha and BLAP75. Enhancement of this BLM activity requires both Topo IIIalpha and BLAP75. Importantly, Topo IIIalpha cannot be substituted by Escherichia coli Top3, and the Holliday junction unwinding activity of BLM-related helicases WRN and RecQ is likewise impervious to Topo IIIalpha and BLAP75. However, the topoisomerase activity of Topo IIIalpha is dispensable for the enhancement of the DNA unwinding reaction. We have also ascertained the requirement for the BLM ATPase activity in double Holliday junction dissolution and DNA unwinding by constructing, purifying, and characterizing specific mutant variants that lack this activity. These results provide valuable information concerning how the functional integrity of the BTB complex is governed by specific protein-protein interactions among the components of this complex and the enzymatic activities of BLM and Topo IIIalpha.

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.

Objective: To evaluate an association of Bloom syndrome (BS) phenotype with an in vivo prooxidant state., Methods: The following endpoints were measured in 4 BS patients, their 6 parents, and 78 controls: a) leukocyte and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG); b) blood glutathione (GSSG and GSH), c) plasma levels of some plasma antioxidants (uric acid, UA, ascorbic acid, AA, alpha- and gamma-tocopherol), and of glyoxal (Glx) and methylglyoxal (MGlx)., Results: Leukocyte 8-OHdG levels were significantly increased in the 4 BS patients vs. 40 controls (p=0.04), while the urinary 8-OHdG levels were non-significantly increased in BS patients. Glutathione disulfide levels and GSSG/GSH ratio were significantly decreased in BS patients vs. 44 controls (p=0.02). The plasma levels of UA in BS patients were significantly increased vs. 24 controls (p=0.005). No significant alterations were found in the in the plasma levels of Glx, MGlx, AA, and tocopherol. No changes in the tested parameters were found in the BS heterozygotes., Conclusion: This report shows a significant increase in oxidative DNA damage in leukocytes and in plasma UA levels from 4 BS patients. Should these data be confirmed in more extensive BS patient groups, an involvement of oxidative stress in the clinical BS phenotype might be suggested.

The RecQ helicases belong to the Superfamily II group of DNA helicases, and are defined by amino acid motifs that show sequence similarity to the catalytic domain of Escherichia coli RecQ. RecQ helicases have crucial roles in the maintenance of genome stability. In humans, there are five RecQ helicases and deficiencies in three of them cause genetic disorders characterised by cancer predisposition, premature aging and/or developmental abnormalities. RecQ helicase-deficient cells exhibit aberrant genetic recombination and/or DNA replication, which result in chromosomal instability and a decreased potential for proliferation. Here, we review the current knowledge of the molecular genetics of RecQ helicases, focusing on the human RecQ helicase disorders and mouse models of these conditions.

Genomic DNA is particularly vulnerable to mutation during S-phase when the two strands of parental duplex DNA are separated during the process of semi-conservative DNA replication. Lesions that are normally repaired efficiently in the context of double stranded DNA can cause replication forks to stall or, more dangerously, collapse. Cells from Bloom's syndrome patients, that lack the RecQ helicase BLM, show defects in the response to replicative stress and contain a multitude of chromosomal aberrations, which primarily arise through excessive levels of homologous recombination. Here, recent findings are reviewed that further our understanding of the role that BLM plays in the management of damaged replication forks.

Spindle assembly checkpoint (SAC) ensures bipolar attachment of chromosomes to the mitotic spindle and is essential for faithful chromosome segregation, thereby preventing chromosome instability (CIN). Genetic evidence suggests a causal link between compromised SAC, CIN, and cancer. Bloom syndrome (BS) is a genetic disorder that predisposes affected individuals to cancer. BS cells exhibit elevated rates of sister chromatid exchange, chromosome breaks, and CIN. The BS gene product, BLM, is a member of the RecQ helicases that are required for maintenance of genome stability. The BLM helicase interacts with proteins involved in DNA replication, recombination, and repair and is required for the repair of stalled-replication forks and in the DNA damage response. Here we present biochemical evidence to suggest a role of BLM phosphorylation during mitosis in maintaining chromosome stability. BLM is associated with the SAC kinase MPS1 and is phosphorylated at S144 in a MPS1-dependent manner. Phosphorylated BLM interacts with polo-like kinase 1, a mitotic kinase that binds to phosphoserine/threonine through its polo-box domain (PBD). Furthermore, BS cells expressing BLM-S144A show normal levels of sister chromatid exchange but fail to maintain the mitotic arrest when SAC is activated and exhibit a broad distribution of chromosome numbers. We propose that MPS1-dependent BLM phosphorylation is important for ensuring accurate chromosome segregation, and its deregulation may contribute to cancer.