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James German's work to establish the natural history and cancer risk associated with Bloom syndrome (BS) has had a strong influence on the generation of scientists and clinicians working to understand other RECQ deficiencies and heritable cancer predisposition syndromes. I summarize work by us and others below, inspired by James German's precedents with BS, to understand and compare BS with the other heritable RECQ deficiency syndromes with a focus on Werner syndrome (WS). What we know, unanswered questions and new opportunities are discussed, as are potential ways to treat or modify WS-associated disease mechanisms and pathways.

Tumor suppressor genes are involved in maintaining genome integrity during reproduction (e.g., meiosis). Thus, deleterious alleles in tumor suppressor-deficient mice would exhibit higher mortality during the perinatal period. A recent aging model proposes that perinatal mortality and age-related deleterious changes might define lifespan. This study aimed to quantitatively understand the relationship between reproduction and lifespan using three established tumor suppressor gene (p53, APC, and RECQL4)-deficient mouse strains with the same C57BL/6 background. Transgenic mice delivered slightly reduced numbers of 1st pups than wild-type mice [ratio: 0.81-0.93 (p = 0.1-0.61)] during a similar delivery period, which suggest that the tumor suppressor gene-deficient mice undergo relatively stable reproduction. However, the transgenic 1st pups died within a few days after birth, resulting in a further reduction in litter size at 3 weeks after delivery compared with that of wild-type mice [ratio: 0.35-0.68 (p = 0.034-0.24)] without sex differences, although the lifespan was variable. Unexpectedly, the significance of reproductive reduction in transgenic mice was decreased at the 2nd or later delivery. Because mice are easily affected by environmental factors, our data underscore the importance of defining reproductive ability through experiments on aging-related reproduction that can reveal a trade-off between fecundity and aging and identify RECQL4 as a novel pleiotropic gene.

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2., Competing Interests: The authors have declared that they have no competing interests.

RecQL4 has been shown to be involved in DNA replication and repair, but its role in DNA damage checkpoint pathway has not been reported. Here, we show that RecQL4 plays an important role in the activation of ataxia telangiectasia mutated (ATM)-dependent checkpoint pathway in human cells. Cells depleted with RecQL4 or Rothmund-Thomson syndrome cells showed significant impairment in the activation of ATM and the downstream effector proteins such as checkpoint kinase 2 and p53 after DNA damage. This defect was recovered with the expression of wild type RecQL4 but not any mutant RecQL4 proteins with defective helicase activities. While RecQL4 failed to show any direct interaction with ATM, it stably interacted with the Mre11-Rad50-Nbs1 complex that is essential for the activation of ATM and was localized on the DNA damage foci. Thus, our results suggest that the helicase activity of RecQL4 plays an important role in the activation of ATM-dependent checkpoint pathway against DNA double strand breaks in human cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Synthetic lethality is a molecular-targeted therapy for selective killing of cancer cells. We exploited a lethal interaction between superoxide dismutase 1 inhibition and Bloom syndrome gene product (BLM) defect for the treatment of colorectal cancer (CRC) cells (HCT 116) with a customized lung cancer screen-1-loaded nanocarrier (LCS-1-NC). The drug LCS-1 has poor aqueous solubility. To overcome its limitations, a customized NC, composed of a magnetite core coated with three polymeric shells, namely, aminocellulose (AC), branched poly(amidoamine), and paraben-PEG, was developed for encapsulating LCS-1. Encapsulation efficiency and drug loading were found to be 74% and 8.2%, respectively. LCS-1-NC exhibited sustained release, with ∼85% of drug release in 24 h. Blank NC (0.5 mg/mL) exhibited cytocompatibility toward normal cells, mainly due to the AC layer. LCS-1-NC demonstrated high killing selectivity (104 times) toward BLM-deficient HCT 116 cells over BLM-proficient HCT 116 cells. Due to enhanced efficacy of the drug using NC, the sensitivity difference for BLM-deficient cells increased to 1.7 times in comparison to that with free LCS-1. LCS-1-NC induced persistent DNA damage and apoptosis, which demonstrates that LCS-1-NC effectively and preferentially killed BLM-deficient CRC cells. This is the first report on the development of a potential drug carrier to improve the therapeutic efficacy of LCS-1 for specific killing of CRC cells having BLM defects.

'BRCAness' is a term used to describe cancer cells that behave similarly to tumors with BRCA1 or BRCA2 mutations. The BRCAness phenotype is associated with hypersensitivity to chemotherapy agents including PARP inhibitors, which are a promising class of recently-licensed anti-cancer treatments. This hypersensitivity arises because of a deficiency in the homologous recombination (HR) pathway for DNA double-strand break repair. To gain further insight into how genetic modifiers of HR contribute to the BRCAness phenotype, we created a new mouse model of BRCAness by generating mice that are deficient in BLM helicase and the Exo1 exonuclease, which are involved in the early stages of HR. We find that cells lacking BLM and Exo1 exhibit a BRCAness phenotype, with diminished HR, and hypersensitivity to PARP inhibitors. We further tested how 53BP1, an important regulator of HR, affects repair efficiency in our BRCAness model. We find that deletion of 53BP1 can relieve several of the repair deficiencies observed in cells lacking BLM and Exo1, just as it does in cells lacking BRCA1. These results substantiate the importance of BRCAness as a concept for classification of cancer cases, and further clarify the role of 53BP1 in regulation of DNA repair pathway choice in mammalian cells.

The BLM gene product, BLM, is a RECQ helicase that is involved in DNA replication and repair of DNA double-strand breaks by the homologous recombination (HR) pathway. During HR, BLM has both pro- and anti-recombinogenic activities, either of which may contribute to maintenance of genomic integrity. We find that in cells expressing a mutant version of BRCA1, an essential HR factor, ablation of BLM rescues genomic integrity and cell survival in the presence of DNA double-strand breaks. Improved genomic integrity in these cells is linked to a substantial increase in the stability of RAD51 at DNA double-strand break sites and in the overall efficiency of HR. Ablation of BLM also rescues RAD51 foci and HR in cells lacking BRCA2 or XRCC2. These results indicate that the anti-recombinase activity of BLM is of general importance for normal retention of RAD51 at DNA break sites and regulation of HR., (© 2017 Patel et al.)

Bloom syndrome (BS), an autosomal recessive disorder of the BLM gene, predisposes sufferers to various cancers. To investigate the mutator phenotype and genetic consequences of DNA double-strand breaks (DSBs) in BS cells, we developed BLM helicase-deficient human cells by disrupting the BLM gene. Cells with a loss of heterozygosity (LOH) due to homologous recombination (HR) or nonhomologous end joining (NHEJ) can be restored with or without site-directed DSB induction. BLM cells exhibited a high frequency of spontaneous interallelic HR with crossover, but noncrossover events with long-tract gene conversions also occurred. Despite the highly interallelic HR events, BLM cells predominantly produced hemizygous LOH by spontaneous deletion. These phenotypes manifested during repair of DSBs. Both NHEJ and HR appropriately repaired DSBs in BLM cells, resulting in hemizygous and homozygous LOHs, respectively. However, the magnitude of the LOH was exacerbated in BLM cells, as evidenced by large deletions and long-tract gene conversions with crossover. BLM helicase suppresses the elongation of branch migration and crossover of double Holliday junctions (HJs) during HR repair, and a deficiency in this enzyme causes collapse, abnormal elongation, and/or preferable resolution to crossover of double HJs, resulting in a large-scale LOH. This mechanism underlies the predisposition for cancer in BS., (Copyright © 2016 Suzuki et al.)

Mutation of Bloom helicase (BLM) causes Bloom syndrome (BS), a rare human genetic disorder associated with genome instability, elevation of sister chromatid exchanges, and predisposition to cancer. Deficiency in BLM homologs in Drosophila and yeast brings about significantly increased rates of recombination between imperfectly matched sequences ("homeologous recombination," or HeR). To assess whether BLM deficiency provokes an increase in HeR in human cells, we transfected an HeR substrate into a BLM-null cell line derived from a BS patient. The substrate contained a thymidine kinase (tk)-neo fusion gene disrupted by the recognition site for endonuclease I-SceI, as well as a functional tk gene to serve as a potential recombination partner for the tk-neo gene. The two tk sequences on the substrate displayed 19% divergence. A double-strand break was introduced by expression of I-SceI and repair events were recovered by selection for G418-resistant clones. Among 181 events recovered, 30 were accomplished via HeR with the balance accomplished by nonhomologous end-joining. The frequency of HeR events in the BS cells was elevated significantly compared to that seen in normal human fibroblasts or in BS cells complemented for BLM expression. We conclude that BLM deficiency enables HeR in human cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aim: To investigate the effects of Recql5 deficiency on liver injury induced by lipopolysaccharide/D-galactosamine (LPS/D-Gal)., Methods: Liver injury was induced in wild type (WT) or Recql5-deficient mice using LPS/D-Gal, and assessed by histological, serum transaminases, and mortality analyses. Hepatocellular apoptosis was quantified by transferase dUTP nick end labeling assay and Western blot analysis of cleaved caspase-3. Liver inflammatory chemokine and cytochrome P450 expression was analyzed by quantitative reverse transcription-PCR. Neutrophil infiltration was evaluated by myeloperoxidase activity. Expression and phosphorylation of ERK, JNK, p65, and H2A.X was determined by Western blot. Oxidative stress was evaluated by measuring malondialdehyde production and nitric oxide synthase, superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase activity., Results: Following LPS/D-Gal exposure, Recql5-deficient mice exhibited enhanced liver injury, as evidenced by more severe hepatic hemorrhage, higher serum aspartate transaminase and alanine transaminase levels, and lower survival rate. As compared to WT mice, Recql5-deficient mice showed an increased number of apoptotic hepatocytes and higher cleaved caspase-3 levels. Recql5-deficient mice exhibited increased DNA damage, as evidenced by increased γ-H2A.X levels. Inflammatory cytokine levels, neutrophil infiltration, and ERK phosphorylation were also significantly increased in the knockout mice. Additionally, Recql5-deficient mice exhibited increased malondialdehyde production and elevated inducible nitric oxide synthase, superoxide dismutase, glutathione peroxidase, catalase, and glutathione reductase activity, indicative of enhanced oxidative stress. Moreover, CYP450 expression was significantly downregulated in Recql5-deficient mice after LPS/D-Gal treatment., Conclusion: Recql5 protects the liver against LPS/D-Gal-induced injury through suppression of hepatocyte apoptosis and oxidative stress and modulation of CYP450 expression.

Formaldehyde is a reactive aldehyde that has been classified as a class I human carcinogen by the International Agency for Cancer Research. There are growing concerns over the possible adverse health effects related to the occupational and environmental human exposures to formaldehyde. Although formaldehyde-induced DNA and protein adducts have been identified, the genomic instability mechanisms and the cellular tolerance pathways associated with formaldehyde exposure are not fully characterized. This study specifically examines the role of a genome stability protein, Bloom (BLM) in limiting formaldehyde-induced cellular and genetic abnormalities. Here, we show that in the absence of BLM protein, formaldehyde-treated cells exhibited increased cellular sensitivity, an immediate cell cycle arrest, and an accumulation of chromosome radial structures. In addition, live-cell imaging experiments demonstrated that formaldehyde-treated cells are dependent on BLM for timely segregation of daughter cells. Both wild-type and BLM-deficient formaldehyde-treated cells showed an accumulation of 53BP1 and γH2AX foci indicative of DNA double-strand breaks (DSBs); however, relative to wild-type cells, the BLM-deficient cells exhibited delayed repair of formaldehyde-induced DSBs. In response to formaldehyde exposure, we observed co-localization of 53BP1 and BLM foci at the DSB repair site, where ATM-dependent accumulation of formaldehyde-induced BLM foci occurred after the recruitment of 53BP1. Together, these findings highlight the significance of functional interactions among ATM, 53BP1, and BLM proteins as responders associated with the repair and tolerance mechanisms induced by formaldehyde., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The 5 known RecQ helicases in humans (RECQ1, BLM, WRN, RECQL4, and RECQ5) have demonstrated roles in diverse genome maintenance mechanisms but their functions in safeguarding the genome from environmental toxicants are poorly understood. Here, we have evaluated a potential role of WRN (mutated in Werner syndrome) and RECQ1 (the most abundant homolog of WRN) in hydroquinone (HQ)- and benzo[a]pyrene (BaP)-induced genotoxicity. Silencing of WRN or RECQ1 expression in HeLa cells increased their sensitivity to HQ and BaP but elicited distinct DNA damage response. The RECQ1-depleted cells exhibited increased replication protein A phosphorylation, Chk1 activation, and DNA double-strand breaks (DSBs) as compared to control or WRN-depleted cells following exposure to BaP treatment. The BaP-induced DSBs in RECQ1-depleted cells were dependent on DNA-dependent protein kinase activity. Notably, loss of WRN in RECQ1-depleted cells ameliorated BaP toxicity. Collectively, our results provide first indication of nonredundant participation of WRN and RECQ1 in protection from the potentially carcinogenic effects of BaP and HQ., (© The Author(s) 2014.)

Mutations within the gene encoding the DNA helicase RECQL4 underlie the autosomal recessive cancer-predisposition disorder Rothmund-Thomson syndrome, though it is unclear how these mutations lead to disease. Here, we demonstrated that somatic deletion of Recql4 causes a rapid bone marrow failure in mice that involves cells from across the myeloid, lymphoid, and, most profoundly, erythroid lineages. Apoptosis was markedly elevated in multipotent progenitors lacking RECQL4 compared with WT cells. While the stem cell compartment was relatively spared in RECQL4-deficent mice, HSCs from these animals were not transplantable and even selected against. The requirement for RECQL4 was intrinsic in hematopoietic cells, and loss of RECQL4 in these cells was associated with increased replicative DNA damage and failed cell-cycle progression. Concurrent deletion of p53, which rescues loss of function in animals lacking the related helicase BLM, did not rescue BM phenotypes in RECQL4-deficient animals. In contrast, hematopoietic defects in cells from Recql4Δ/Δ mice were fully rescued by a RECQL4 variant without RecQ helicase activity, demonstrating that RECQL4 maintains hematopoiesis independently of helicase activity. Together, our data indicate that RECQL4 participates in DNA replication rather than genome stability and identify RECQL4 as a regulator of hematopoiesis with a nonredundant role compared with other RecQ helicases.

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double-strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short-term rapamycin treatment. Long-term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near-normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS., (© 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund-Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated β-galactosidase (SA-β-gal), higher expression of p16(INK4a) or/and p21(WAF1) and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4(HD)). Tail fibroblasts from Recql4(HD) showed increased SA-β-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4(HD) mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.

A major medical challenge in the elderly is osteoporosis and the high risk of fracture. Telomere dysfunction is a cause of cellular senescence and telomere shortening, which occurs with age in cells from most human tissues, including bone. Telomere defects contribute to the pathogenesis of two progeroid disorders characterized by premature osteoporosis, Werner syndrome and dyskeratosis congenital. It is hypothesized that telomere shortening contributes to bone aging. We evaluated the skeletal phenotypes of mice with disrupted telomere maintenance mechanisms as models for human bone aging, including mutants in Werner helicase (Wrn(-/-)), telomerase (Terc(-/-)) and Wrn(-/-)Terc(-/-) double mutants. Compared with young wild-type (WT) mice, micro-computerized tomography analysis revealed that young Terc(-/-) and Wrn(-/-)Terc(-/-) mice have decreased trabecular bone volume, trabecular number and trabecular thickness, as well as increased trabecular spacing. In cortical bone, young Terc(-/-) and Wrn(-/-)Terc(-/-) mice have increased cortical thinning, and increased porosity relative to age-matched WT mice. These trabecular and cortical changes were accelerated with age in Terc(-/-) and Wrn(-/-)Terc(-/-) mice compared with older WT mice. Histological quantification of osteoblasts in aged mice showed a similar number of osteoblasts in all genotypes; however, significant decreases in osteoid, mineralization surface, mineral apposition rate and bone formation rate in older Terc(-/-) and Wrn(-/-)Terc(-/-) bone suggest that osteoblast dysfunction is a prominent feature of precocious aging in these mice. Except in the Wrn(-/-) single mutant, osteoclast number did not increase in any genotype. Significant alterations in mechanical parameters (structure model index, degree of anistrophy and moment of inertia) of the Terc(-/-) and Wrn(-/-)Terc(-/-) femurs compared with WT mice were also observed. Young Wrn(-/-)Terc(-/-) mice had a statistically significant increase in bone-marrow fat content compared with young WT mice, which remained elevated in aged double mutants. Taken together, our results suggest that Terc(-/-) and Wrn(-/-)Terc(-/-) mutants recapitulate the human bone aging phenotype and are useful models for studying age-related osteoporosis.

Werner syndrome (WS) results from dysfunction of the WRN protein, and is associated with premature aging and early death. Here we report that loss of WRN function elicits accumulation of the Yes-associated protein (YAP protein), a major effector of the Hippo tumor suppressor pathway, both experimentally and in WS-derived fibroblasts. YAP upregulation correlates with slower cell proliferation and accelerated senescence, which are partially mediated by the formation of a complex between YAP and the PML protein, whose activity promotes p53 activation. The ATM kinase is necessary for YAP and PML accumulation in WRN-depleted cells. Notably, the depletion of either YAP or PML partially impairs the induction of senescence following WRN loss. Altogether, our findings reveal that loss of WRN activity triggers the activation of an ATM-YAP-PML-p53 axis, thereby accelerating cellular senescence. The latter has features of SASP (senescence-associated secretory phenotype), whose protumorigenic properties are potentiated by YAP, PML and p53 depletion.

Breast cancer occur both in hereditary and sporadic forms, and the later one comprises an overwhelming majority of breast cancer cases among women. Numerical and structural alterations involving chromosome 8, with loss of short arm (8p) and gain of long arm (8q), are frequently observed in breast cancer cells and tissues. In this study, we show that most of the human breast tumor cell lines examined display an over representation of 8q24, a chromosomal locus RecQL4 is regionally mapped to, and consequently, a markedly elevated level of RecQL4 expression. An increased RecQL4 mRNA level was also observed in a majority of clinical breast tumor samples (38/43) examined. shRNA-mediated RecQL4 suppression in MDA-MB453 breast cancer cells not only significantly inhibit the in vitro clonogenic survival and in vivo tumorigenicity. Further studies demonstrate that RecQL4 physically interacts with a major survival factor-survivin and its protein level affects survivin expression. Although loss of RecQL4 function due to gene mutations causally linked to occurrence of human RTS with features of premature aging and cancer predisposition, our studies provide the evidence that overexpression of RecQL4 due to gene amplification play a critical role in human breast tumor progression.

Human RECQL5 is a member of the RecQ helicase family, which maintains genome stability via participation in many DNA metabolic processes, including DNA repair. Human cells lacking RECQL5 display chromosomal instability. We find that cells depleted of RECQL5 are sensitive to oxidative stress, accumulate endogenous DNA damage, and increase the cellular poly(ADP-ribosyl)ate response. In contrast to the RECQ helicase family members WRN, BLM, and RECQL4, RECQL5 accumulates at laser-induced single-strand breaks in normal human cells. RECQL5 depletion affects the levels of PARP-1 and XRCC1, and our collective results suggest that RECQL5 modulates and/or directly participates in base excision repair of endogenous DNA damage, thereby promoting chromosome stability in normal human cells.

We undertook genetic and nongenetic approaches to investigate the relationship between telomere maintenance and osteoblast differentiation, as well as to uncover a possible link between a known mediator of cellular aging and senile bone loss. Using mouse models of disrupted telomere maintenance molecules, including mutants in the Werner helicase (Wrn(-/-) ), telomerase (Terc(-/-) ), and Wrn(-/-) Terc(-/-) double mutants predisposed to accelerated bone loss, we measured telomere dysfunction-induced foci (TIFs) and markers of osteoblast differentiation in mesenchymal progenitor cells (MPCs). We found that telomere maintenance is directly and significantly related to osteoblast differentiation, with dysfunctional telomeres associated with impaired differentiation independent of proliferation state. Telomere-mediated defects in osteoblast differentiation are associated with increased p53/p21 expression and concomitant reduction in RUNX2. Conversely, MPCs from p53(-/-) mice do not have substantial telomere dysfunction and spontaneously differentiate into osteoblasts. These results suggest that critical telomere dysfunction may be a prominent mechanism for age-related osteoporosis and limits MPC differentiation into bone-forming cells via the p53/p21 pathway., (© 2012 The Authors. Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

DNA Polymerase δ (Pol δ) and the Werner syndrome protein, WRN, are involved in maintaining cellular genomic stability. Pol δ synthesizes the lagging strand during replication of genomic DNA and also functions in the synthesis steps of DNA repair and recombination. WRN is a member of the RecQ helicase family, loss of which results in the premature aging and cancer-prone disorder, Werner syndrome. Both Pol δ and WRN encode 3' → 5' DNA exonuclease activities. Pol δ exonuclease removes 3'-terminal mismatched nucleotides incorporated during replication to ensure high fidelity DNA synthesis. WRN exonuclease degrades DNA containing alternate secondary structures to prevent formation and enable resolution of stalled replication forks. We now observe that similarly to WRN, Pol δ degrades alternate DNA structures including bubbles, four-way junctions, and D-loops. Moreover, WRN and Pol δ form a complex with enhanced ability to hydrolyze these structures. We also present evidence that WRN can proofread for Pol δ; WRN excises 3'-terminal mismatches to enable primer extension by Pol δ. Consistent with our in vitro observations, we show that WRN contributes to the maintenance of DNA synthesis fidelity in vivo. Cells expressing limiting amounts (∼10% of normal) of WRN have elevated mutation frequencies compared with wild-type cells. Together, our data highlight the importance of WRN exonuclease activity and its cooperativity with Pol δ in preserving genome stability, which is compromised by the loss of WRN in Werner syndrome.

MYC-induced DNA damage is exacerbated in WRN-deficient cells, leading to replication stress and accelerated cellular senescence. To determine whether WRN deficiency impairs MYC-driven tumor development, we used both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non-small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knockdown was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (Wrn(Δhel/Δhel)) resulted in a significant delay in emergence of lethal lymphomas, extending tumor-free survival by more than 30%. Analysis of preneoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc Wrn(Δhel/Δhel) lymphomas showed a marked increase in expression of the CDK inhibitor, p16(Ink4a), as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies show that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in preneoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor-free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers.

In recBCD sbcB sbcC(D) mutants of Escherichia coli homologous recombination proceeds via RecF pathway, which is thought to require RecQ, UvrD and HelD helicases at its initial stage. It was previously suggested that depletion of all three helicases totally abolishes the RecF pathway. The present study (re)examines the roles of these helicases in transductional recombination, and in recombinational repair of UV-induced DNA damage in the RecF pathway. The study has employed the ΔrecBCD ΔsbcB sbcC201 and ΔrecBCD sbcB15 sbcC201 strains, carrying combinations of mutations in recQ, uvrD, and helD genes. We show that in ΔrecBCD ΔsbcB sbcC201 strains, recombination requires exclusively the RecQ helicase. In ΔrecBCD sbcB15 sbcC201 strains, RecQ may be partially substituted by UvrD helicase. The HelD helicase is dispensable for recombination in both backgrounds. Our results also suggest that significant portion of recombination events in the RecF pathway is independent of RecQ, UvrD and HelD. These events are initiated either by RecJ nuclease alone or by RecJ nuclease associated with an unknown helicase. Inactivation of exonuclease VII by a xseA mutation further decreases the requirement for helicase activity in the RecF pathway. We suggest that elimination of nucleases acting on 3' single-strand DNA ends reduces the necessity for helicases in initiation of recombination., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.

p16(Ink4a) is a tumor suppressor and a marker for cellular senescence. Previous studies have shown that p16(Ink4a) plays an important role in the response to DNA damage signals caused by telomere dysfunction. In this study, we crossed Wrn(-/-) and p16(Ink4a-/-) mice to knock out the p16(Ink4a) function in a Wrn null background. Growth curves showed that loss of p16(Ink4a) could rescue the growth barriers that are observed in Wrn(-/-) mouse embryonic fibroblasts (MEFs). By challenging the MEFs with the global genotoxin doxorubicin, we showed that loss of p16(Ink4a) did not dramatically affect the global DNA damage response of Wrn(-/-) MEFs induced by doxorubicin. However, in response to telomere dysfunction initiated by the telomere damaging protein TRF2(ΔBΔM), loss of p16(Ink4a) could partially overcome the DNA damage response by disabling p16(Ink4a) up-regulation and reducing the accumulation of γ-H2AX that is observed in Wrn(-/-) MEFs. Furthermore, in response to TRF2(ΔBΔM) overexpression, Wrn(-/-) MEFs senesced within several passages. In contrast, p16(Ink4a-/-) and p16(Ink4a-/-)Wrn(-/-) MEFs could continuously grow and lose expression of the exogenous TRF2(ΔBΔM) in their late passages. In summary, our data suggest that in the context of telomere dysfunction, loss of p16(Ink4a) function could prevent cells from senescence. These results shed light on the anti-aging strategy through regulation of p16(Ink4a) expression.

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.

Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.

Drosophila melanogaster RecQ5, a member of the RecQ family, is expressed in early embryos. The loss of maternally-derived RecQ5 leads to spontaneous mitotic defects in syncytial embryos. We demonstrate that the mitotic defects are derived from anaphase DNA bridges. Pairs of daughter nuclei that had been linked by the bridges concurrently exited from the cycle and were eliminated by Chk2-dependent centrosome inactivation. These results suggest that the lack of RecQ5 leads to spontaneous double-stranded DNA breaks (DSBs). RecQ5 may function in the resolution of anaphase DNA bridges during mitosis or in DSB repair during interphase in syncytial Drosophila embryos., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The conserved RECQ5 DNA helicase is a tumor suppressor in mammalian cells. Defects in RECQ5 lead to the accumulation of spontaneous DNA double-stranded breaks (DSBs) during replication, despite the fact that these cells are proficient in DSB repair by homologous recombination (HR). The reason for this is unknown. Here, we demonstrate that these DSBs are linked to RNA polymerase II (RNAPII)-dependent transcription. In human RECQ5-depleted cells, active RNAPII accumulates on chromatin, and DNA breaks are associated with an RNAPII-dependent transcribed locus. Hence, transcription inhibition eliminates both active RNAPII and spontaneous DSB formation. In addition, the regulatory effect of RECQ5 on transcription and its interaction with RNAPII are enhanced in S-phase cells, supporting a role for RECQ5 in preventing transcription-associated DSBs during replication. Finally, we show that the SET2-RPB1 interaction (SRI) domain of human RECQ5 is important for suppressing spontaneous DSBs and the p53-dependent transcription stress response caused by the stalling of active RNAPII on DNA. Thus, our studies provide novel insights into a mechanism by which RECQ5 regulates the transcription machinery via its dynamic interaction with RNAPII, thereby preventing genome instability.

In somatic cells, Holliday junctions can be formed between sister chromatids during the recombinational repair of DNA breaks or after replication fork demise. A variety of processes act upon Holliday junctions to remove them from DNA, in events that are critical for proper chromosome segregation. In human cells, the BLM protein, inactivated in individuals with Bloom's syndrome, acts in combination with topoisomerase IIIα, RMI1 and RMI2 (BTR complex) to promote the dissolution of double Holliday junctions. Cells defective for BLM exhibit elevated levels of sister chromatid exchanges (SCEs) and patients with Bloom's syndrome develop a broad spectrum of early-onset cancers caused by chromosome instability. MUS81-EME1 (refs 4-7), SLX1-SLX4 (refs 8-11) and GEN1 (refs 12, 13) also process Holliday junctions but, in contrast to the BTR complex, do so by endonucleolytic cleavage. Here we deplete these nucleases from Bloom's syndrome cells to analyse human cells compromised for the known Holliday junction dissolution/resolution pathways. We show that depletion of MUS81 and GEN1, or SLX4 and GEN1, from Bloom's syndrome cells results in severe chromosome abnormalities, such that sister chromatids remain interlinked in a side-by-side arrangement and the chromosomes are elongated and segmented. Our results indicate that normally replicating human cells require Holliday junction processing activities to prevent sister chromatid entanglements and thereby ensure accurate chromosome condensation. This phenotype was not apparent when both MUS81 and SLX4 were depleted from Bloom's syndrome cells, suggesting that GEN1 can compensate for their absence. Additionally, we show that depletion of MUS81 or SLX4 reduces the high frequency of SCEs in Bloom's syndrome cells, indicating that MUS81 and SLX4 promote SCE formation, in events that may ultimately drive the chromosome instabilities that underpin early-onset cancers associated with Bloom's syndrome.

Werner syndrome (WS) is a rare disorder characterized by the premature onset of several pathologies associated with aging. The gene responsible for WS codes for a RecQ-type DNA helicase and is believed to be involved in different aspects of DNA repair, replication, and transcription. We recently identified the Scaffold attachment factor B1 (SAFB1) as a potential interactants in human cells. SAFB1 is a multifunctional protein that binds both nucleic acids and is involved in the attachment of chromatin to the nuclear matrix, transcription, and stress response. Mice lacking SAFB1 exhibit developmental abnormalities in their lungs, high incidence of perinatal lethality, and adults develop different types of tumors. Mouse embryonic fibroblasts from Safb1-null animals are immortalized in culture. In this study, mice with a mutation in the helicase domain of the Wrn gene were crossed to Safb1-null mice. Double homozygous mutant mice exhibited increased apoptosis, a lower cell proliferation rate in their lungs and a higher incidence of perinatal death compared to Safb1-null mice. Few double homozygous mutants survived weaning and died before the age of six months. Finally, mouse embryonic fibroblasts lacking a functional Wrn helicase inhibited the immortalization of Safb1-null cells. These results indicate that an intact Wrn protein is required for immortalization and tumorigenesis in Safb1-null mice.

Background: Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis., Methodology/principal Findings: We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges., Conclusions/significance: Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency.

Loss-of-function mutations in the human RecQ helicase genes WRN and BLM respectively cause the genetic instability/cancer predisposition syndromes Werner syndrome and Bloom syndrome. To identify common and unique functions of WRN and BLM, we systematically analyzed cell proliferation, cell survival, and genomic damage in isogenic cell lines depleted of WRN, BLM, or both proteins. Cell proliferation and survival were assessed before and after treatment with camptothecin, cis-diamminedichloroplatinum(II), hydroxyurea, or 5-fluorouracil. Genomic damage was assessed, before and after replication arrest, by gamma-H2AX staining, which was quantified at the single-cell level by flow cytometry. Cell proliferation was affected strongly by the extent of WRN and/or BLM depletion, and more strongly by BLM than by WRN depletion (P = 0.005). The proliferation of WRN/BLM-codepleted cells, in contrast, did not differ from BLM-depleted cells (P = 0.34). BLM-depleted and WRN/BLM-codepleted cells had comparably impaired survival after DNA damage, whereas WRN-depleted cells displayed a distinct pattern of sensitivity to DNA damage. BLM-depleted and WRN/BLM-codepleted cells had similar, significantly higher gamma-H2AX induction levels than did WRN-depleted cells. Our results provide new information on the role of WRN and BLM in determining cell proliferation, cell survival, and genomic damage after chemotherapeutic DNA damage or replication arrest. We also provide new information on functional redundancy between WRN and BLM. These results provide a strong rationale for further developing WRN and BLM as biomarkers of tumor chemotherapeutic responsiveness., ((c)2010 AACR.)

Aim: To investigate whether Recql5, a DNA helicase that plays an important role in the maintenance of genome integrity, is a tumor suppressor in the gastrointestinal tract in mice., Methods: We generated cohorts of both Recql5-proficient and Recql5-deficient Apc(min/+) mice and compared the tumor susceptibility in their gastrointestinal tracts., Results: Recql5 deficiency in Apc(min/+) mice resulted in a significant increase in the tumor incidence in both the colon (P = 0.0162) and the small intestine (P < 0.01). These findings have provided the first genetic evidence for a tumor suppression role of Recql5 in the gastrointestinal tract of mice. Importantly, since mouse Recql5 and human RECQL5 are highly conserved, these findings also suggest that RECQL5 may be a tumor suppressor for human colon cancer., Conclusion: Recql5 has a tumor suppression role in the mouse gastrointestinal tract.

Bloom's syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS patients, is one of five mammalian RecQ helicases. Although BLM has been shown to promote genome stability by assisting in the repair of DNA structures that arise during homologous recombination in somatic cells, less is known about its role in meiotic recombination primarily because of the embryonic lethality associated with Blm deletion. However, the localization of BLM protein on meiotic chromosomes together with evidence from yeast and other organisms implicates a role for BLM helicase in meiotic recombination events, prompting us to explore the meiotic phenotype of mice bearing a conditional mutant allele of Blm. In this study, we show that BLM deficiency does not affect entry into prophase I but causes severe defects in meiotic progression. This is exemplified by improper pairing and synapsis of homologous chromosomes and altered processing of recombination intermediates, resulting in increased chiasmata. Our data provide the first analysis of BLM function in mammalian meiosis and strongly argue that BLM is involved in proper pairing, synapsis, and segregation of homologous chromosomes; however, it is dispensable for the accumulation of recombination intermediates.

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.

The mus309 gene in Drosophila melanogaster encodes a RecQ helicase which is involved in DNA double-strand break (DSB) repair. In a brood pattern analysis, it was observed that in mus309 mutant females, the frequency of single crossovers in the central cv-v interval of the X chromosome was reduced in young females but returned to the level of the wild type control as the females aged. In the proximal v-f interval, the frequency of single crossovers was increased during the entire experimental period. In particular, it was observed that the frequency of double crossovers, as well as the coefficient of coincidence first increased but then gradually decreased, finally reaching the level of the control flies, as the females aged. Map distances increased due to the mus309 mutation in both gene interval studies, but they did not change as the females aged, a result suggesting that the mus309 gene controls the distribution of DSBs to be repaired as crossovers instead of non-crossovers. The results suggest a mechanism for the centromere effect of crossing over in Drosophila, viz the fact the frequency of meiotic crossing over reduces with the age of the female, and that the reduction is more pronounced the closer the interval is to the proximal heterochromatin of the chromosome arm. According to the model suggested, the centromere effect is simply a matter of the balance between different pathways of the repair of the DSBs of DNA.

Mutations in the BLM gene cause human Bloom syndrome (BS), an autosomal recessive disorder of growth retardation, immunodeficiency and cancer predisposition. Homozygous null Blm(m3/m3) mice are cancer prone with a 5-fold increased risk of cancer compared with Blm(m3/+) and Blm(+/+) mice. Irradiation of Blm(m3/m3) mice increased the risk to 28-fold. Tumors occurred mainly in the hematopoietic system and were similar to those in BS based on detailed histologic and immunohistochemical analyses. Irradiated Blm-deficient mice thus provide a novel model for understanding accelerated malignancies in BS and a new platform for investigating the molecular basis for a wide range of hematopoietic neoplasms., (Published by Elsevier Ltd.)

Werner's syndrome (WS) is a rare human autosomal recessive segmental progeroid syndrome clinically characterized by atherosclerosis, cancer, osteoporosis, type 2 diabetes mellitus and ocular cataracts. The WRN gene codes for a RecQ helicase which is present in many tissues. Although the exact functions of the WRN protein remain unclear, accumulating evidence suggests that it participates in DNA repair, replication, recombination and telomere maintenance. It has also been proposed that WRN participates in RNA polymerase II-dependent transcription. However no promoter directly targeted by WRN has yet been identified. In this work, we report mammalian genes that are WRN targets. The rat CYP2B2 gene and its closely related mouse homolog, Cyp2b10, are both strongly induced in liver by phenobarbital. We found that there is phenobarbital-dependent recruitment of WRN to the promoter of the CYP2B2 gene as demonstrated by chromatin immunoprecipitation analysis. Mice homozygous for a Wrn mutation deleting part of the helicase domain showed a decrease in basal and phenobarbital-induced CYP2B10 mRNA levels compared to wild type animals. The phenobarbital-induced level of CYP2B10 protein was also reduced in the mutant mice. Electrophoretic mobility shift assays showed that WRN can participate in the formation of a complex with a specific sequence within the CYP2B2 basal promoter. Hence, there is a WRN binding site in a region of DNA sequence to which WRN is recruited in vivo. Taken together, these results suggest that WRN participates in transcription of CYP2B genes in liver and identifies the first physical interaction between a specific promoter sequence and WRN.

Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn-/- mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn-/- mouse primary fibroblasts with previous analyses of primary and transformed fibroblasts from WS patients and with newly derived, WRN-depleted human primary fibroblasts. These analyses confirmed previously reported cellular phenotypes of WRN-mutant and WRN-deficient human fibroblasts, and demonstrated that the human WRN-deficient cellular phenotype can be detected in cells grown in 5% or in 20% oxygen. In contrast, we did not identify prominent cellular phenotypes present in WRN-deficient human cells in Wrn-/- mouse fibroblasts. Our results indicate that human and mouse fibroblasts have different functional requirements for WRN protein, and that the absence of a strong cellular phenotype may in part explain the failure of Wrn-/- mice to develop an organismal phenotype resembling Werner syndrome., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Mechanisms of telomere replication remain poorly defined. It has been suggested that G-rich telomeric strand replication by lagging mechanisms requires, in a stochastic way, the WRN protein. Here we show that this requirement is more systematic than previously thought. Our data are compatible with a situation in which, in the absence of WRN, DNA synthesis at replication forks is uncoupled, thus allowing replication to continue on the C strand, while single G strands accumulate. We also show that in cells in which both WRN and POT1 are limiting, both G- and C-rich telomeric strands shorten, suggesting a complete replication block. Under this particular condition, expression of a fragment spanning the two POT1-OB (oligonucleotide-binding) fold domains is able to restore C (but not G) strand replication, suggesting that binding of POT1 to the lagging strand allows DNA synthesis uncoupling in the absence of WRN. Furthermore, in vitro experiments indicate that purified POT1 has a higher affinity for the telomeric G-rich strand than purified RPA. We propose a model in which the relative enrichments of POT1 versus RPA on the telomeric lagging strand allows or does not allow uncoupling of DNA synthesis at the replication fork. Our study reveals an unanticipated role for hPOT1 during telomere replication.

Werner syndrome is a premature aging disorder characterized by cancer predisposition that is caused by loss of the Werner syndrome protein (WRN) helicase/exonuclease DNA repair protein. Hexavalent chromium is an environmental carcinogen and genotoxicant that is associated with respiratory cancers and induces several forms of DNA damage, including lesions that interfere with DNA replication. Based on the evidence that WRN protein facilitates repair of stalled and collapsed replication forks, we hypothesized that WRN functions in the cellular response to and recovery from Cr(VI)-induced genotoxicity and genomic instability. Here we report that human cells deficient in WRN protein are hypersensitive to Cr(VI) toxicity, and exhibit a delayed reduction in DNA breaks and stalled replication forks, indicated by gammaH2AX foci, during recovery from Cr(VI) exposure. Cr(VI)-induced WRN protein translocation from the nucleoli into nucleoplasmic foci in S-phase cells, and these foci colocalized with gammaH2AX foci indicating WRN responds to replication-associated DNA damage. As further evidence that Cr(VI) triggers stalled DNA replication, we observed Cr(VI) treatment induced an accumulation of cells in S-phase that exhibited high levels of gammaH2AX foci. Therefore, these data demonstrate a novel role for WRN protein in cellular protection against the environmental genotoxicant Cr(VI) and further provide evidence that Cr(VI) induces DNA replicative stress which has implications for aging and cancer.

Werner syndrome (WS) is characterized by the premature onset of several age-associated pathologies. The protein deficient in WS (WRN) is a RecQ-type DNA helicase involved in DNA repair, replication, telomere maintenance and transcription. However, precisely how WRN deficiency leads to the numerous WS pathologies is still unknown. Here we use short-term siRNA-based inhibition of WRN to test the direct consequences of its loss on gene expression. Importantly, this short-term knock down of WRN protein level was sufficient to trigger an expression profile resembling fibroblasts established from old donor patients. In addition, this treatment altered sets of genes involved in 14 distinct biological pathways. Besides the already known impact of WRN on DNA replication, DNA repair, the p21/p53 pathway, and cell cycle, gene set enrichment analyses of our microarray data also uncover significant impact on the MYC, E2F, cellular E2A and ETV5 transcription factor pathways as well as adipocyte differentiation, HIF1, NFkappaB and IL-6 pathways. Finally, short-term siRNA-based inhibition of mouse Wrn expression in the pre-adipocyte cell line 3T3-L1 confirmed the impact of WRN on adipogenesis. These results are consistent with the pro-inflammatory status and lipid abnormalities observed in WS patients. This approach thus identified new effectors of WRN activity that might contribute to the WS phenotype.

c-Myc interacts with components of the pre-replication complex and directly regulates DNA replication [1]. However the consequences of this novel c-Myc function on cell cycle dynamics and replication-associated damage are unknown. Here, we show that c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase while c-Myc deficient fibroblasts exhibit a prolonged S-phase. We also show that the Werner DNA helicase protein (WRN) plays a critical role in supporting c-Myc-driven S-phase, as depletion of WRN in c-Myc overexpressing cells increases DNA damage specifically at sites of DNA synthesis. This excess DNA damage activates a "replication stress" pathway involving ATR, CHK1, CHK2, and p53, leading to rapid senescence of WRN deficient c-Myc overexpressing cells. Indeed, depletion of p53 rescues this senescence response. We propose that WRN functions to repair abnormal replication structures caused by the acceleration of DNA replication by c-Myc. This work provides an additional mechanistic explanation for c-Myc-induced DNA damage and senescence, and reveals a vulnerability of c-Myc overexpressing cells that could potentially be exploited in cancer therapy.

Genetic screens performed in model organisms have helped identify key components of the RNA interference (RNAi) pathway. Recessive genetic screens have recently become feasible through the use of mouse embryonic stem (ES) cells that are Bloom's syndrome protein (Blm) deficient. Here, we developed and performed a recessive genetic screen to identify components of the mammalian RNAi pathway in Blm-deficient ES cells. Genome-wide mutagenesis using a retroviral gene trap strategy resulted in the isolation of putative homozygous RNAi mutant cells. Candidate clones were confirmed by an independent RNAi-based reporter assay and the causative gene trap integration site was identified using molecular techniques. Our screen identified multiple mutant cell lines of Argonaute 2 (Ago2), a known essential component of the RNAi pathway. This result demonstrates that true RNAi components can be isolated by this screening strategy. Furthermore, Ago2 homozygous mutant ES cells provide a null genetic background to perform mutational analyses of the Ago2 protein. Using genetic rescue, we resolve an important controversy regarding the role of two phenylalanine residues in Ago2 activity.

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11-13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.

The RecQ family helicase BLM is critically involved in the maintenance of genomic stability, and BLM mutation causes the heritable disorder Bloom's syndrome. Affected individuals suffer from a predisposition to a multitude of cancer types and an ill-defined immunodeficiency involving low serum Ab titers. To investigate its role in B cell biology, we inactivated murine Blm specifically in B lymphocytes in vivo. Numbers of developing B lymphoid cells in the bone marrow and mature B cells in the periphery were drastically reduced upon Blm inactivation. Of the major peripheral B cell subsets, B1a cells were most prominently affected. In the sera of Blm-deficient naive mice, concentrations of all Ig isotypes were low, particularly IgG3. Specific IgG Ab responses upon immunization were poor and mutant B cells exhibited a generally reduced Ab class switch capacity in vitro. We did not find evidence for a crucial role of Blm in the mechanism of class switch recombination. However, a modest shift toward microhomology-mediated switch junction formation was observed in Blm-deficient B cells. Finally, a cohort of p53-deficient, conditional Blm knockout mice revealed an increased propensity for B cell lymphoma development. Impaired cell cycle progression and survival as well as high rates of chromosomal structural abnormalities in mutant B cell blasts were identified as the basis for the observed effects. Collectively, our data highlight the importance of BLM-dependent genome surveillance for B cell immunity by ensuring proper development and function of the various B cell subsets while counteracting lymphomagenesis.

Bloom syndrome is caused by homozygous mutations in BLM, which encodes a RecQ DNA helicase. Patient-derived cells deficient in BLM helicase activity exhibit genetic instability--apparent cytogenetically as sister chromatid exchanges--and activated DNA damage signaling. In this report, we show that BLM-knockout colorectal cancer cells exhibited endogenous, ATM-dependent double-strand DNA break responses similar to those recently observed in Bloom syndrome patient-derived cells. Xenograft tumors established from BLM-deficient cancer cells were not radiosensitive, but exhibited growth impairment that was comparable to that of wild type tumors treated with a single, high dose of ionizing radiation. These results suggest that pharmacological inhibitors of BLM would have a radiomimetic effect and that transient inhibition of BLM activity might be a viable strategy for anticancer therapy.