Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom's syndrome.
ABSTRACT: Bloom's syndrome (BS) and Fanconi anemia (FA) are autosomal recessive disorders characterized by cancer and chromosomal instability. BS and FA group J arise from mutations in the BLM and FANCJ genes, respectively, which encode DNA helicases. In this work, FANCJ and BLM were found to interact physically and functionally in human cells and co-localize to nuclear foci in response to replication stress. The cellular level of BLM is strongly dependent upon FANCJ, and BLM is degraded by a proteasome-mediated pathway when FANCJ is depleted. FANCJ-deficient cells display increased sister chromatid exchange and sensitivity to replication stress. Expression of a FANCJ C-terminal fragment that interacts with BLM exerted a dominant negative effect on hydroxyurea resistance by interfering with the FANCJ-BLM interaction. FANCJ and BLM synergistically unwound a DNA duplex substrate with sugar phosphate backbone discontinuity, but not an 'undamaged' duplex. Collectively, the results suggest that FANCJ catalytic activity and its effect on BLM protein stability contribute to preservation of genomic stability and a normal response to replication stress.
Project description:Genomic instability is a hallmark of cancer and aging. Premature aging (progeroid) syndromes are often caused by mutations in genes whose function is to ensure genomic integrity. The RecQ family of DNA helicases is highly conserved and plays crucial roles as genome caretakers. In humans, mutations in three RecQ genes - BLM, WRN, and RECQL4 - give rise to Bloom's syndrome (BS), Werner syndrome (WS), and Rothmund-Thomson syndrome (RTS), respectively. WS is a prototypic premature aging disorder; however, the clinical features present in BS and RTS do not indicate accelerated aging. The BLM helicase has pivotal functions at the crossroads of DNA replication, recombination, and repair. BS cells exhibit a characteristic form of genomic instability that includes excessive homologous recombination. The excessive homologous recombination drives the development in BS of the many types of cancers that affect persons in the normal population. Replication delay and slower cell turnover rates have been proposed to explain many features of BS, such as short stature. More recently, aberrant transcriptional regulation of growth and survival genes has been proposed as a hypothesis to explain features of BS.
Project description:Bloom's syndrome (BS) is a cancer predisposition disorder caused by mutation of the BLM gene, encoding a member of the RecQ helicase family. Although the phenotype of BS cells is suggestive of a role for BLM in repair of stalled or damaged replication forks, thus far there has been no direct evidence that BLM associates with any of the three human replicative DNA polymerases. Here, we show that BLM interacts specifically in vitro and in vivo with p12, the smallest subunit of human POL delta (hPOL delta). The hPOL delta enzyme, as well as the isolated p12 subunit, stimulates the DNA helicase activity of BLM. Conversely, BLM stimulates hPOL delta strand displacement activity. Our results provide the first functional link between BLM and the replicative machinery in human cells, and suggest that BLM might be recruited to sites of disrupted replication through an interaction with hPOL delta. Finally, our data also define a novel role for the poorly characterized p12 subunit of hPOL delta.
Project description:Bloom's syndrome (BS) which associates genetic instability and predisposition to cancer is caused by mutations in the BLM gene encoding a RecQ family 3'-5' DNA helicase. It has been proposed that the generation of genetic instability in BS cells could result from an aberrant non-homologous DNA end joining (NHEJ), one of the two main DNA double-strand break (DSB) repair pathways in mammalian cells, the second major pathway being homologous recombination (HR). Using cell extracts, we report first that Ku70/80 and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), key factors of the end-joining machinery, and BLM are located in close proximity on DNA and that BLM binds to DNA only in the absence of ATP. In the presence of ATP, BLM is phosphorylated and dissociates from DNA in a strictly DNA-PKcs-dependent manner. We also show that BS cells display, in vivo, an accurate joining of DSBs, reflecting thus a functional NHEJ pathway. In sharp contrast, a 5-fold increase of the HR-mediated DNA DSB repair in BS cells was observed. These results support a model in which NHEJ activation mediates BLM dissociation from DNA, whereas, under conditions where HR is favored, e.g. at the replication fork, BLM exhibits an anti-recombinogenic role.
Project description:Bloom's syndrome (BS) is a rare genetic disorder characterized by a broad range of symptoms and, most importantly, a predisposition to many types of cancers. Cells derived from patients with BS exhibit an elevated rate of somatic recombination and hypermutability, supporting a role for bleomycin (BLM) in the maintenance of genomic integrity. BLM is thought to participate in several DNA transactions, the failure of which could give raise to genomic instability, and to interact with many proteins involved in replication, recombination, and repair. In this study, we show that BLM function is specifically required to properly relocalize the RAD50/MRE11/NBS1 (RMN) complex at sites of replication arrest, but is not essential in the activation of BRCA1 either after stalled replication forks or gamma-rays. We also provide evidence that BLM is phosphorylated after replication arrest in an Ataxia and RAD3-related protein (ATR)-dependent manner and that phosphorylation is not required for subnuclear relocalization. Therefore, in ATR dominant negative mutant cells, the assembly of the RMN complex in nuclear foci after replication blockage is almost completely abolished. Together, these results suggest a relationship between BLM, ATR, and the RMN complex in the response to replication arrest, proposing a role for BLM protein and RMN complex in the resolution of stalled replication forks.
Project description:Bloom's syndrome (BS) is an autosomal recessive disease, caused by mutations in the BLM gene. This gene codes for BLM protein, which is a helicase involved in DNA repair. DNA repair is especially important for the development and maturation of the T and B cells. Since BLM is involved in DNA repair, we aimed to study if BLM deficiency affects T and B cell development and especially somatic hypermutation (SHM) and class switch recombination (CSR) processes. Clinical data of six BS patients was collected, and immunoglobulin serum levels were measured at different time points. In addition, we performed immune phenotyping of the B and T cells and analyzed the SHM and CSR in detail by analyzing IGHA and IGHG transcripts using next-generation sequencing. The serum immunoglobulin levels were relatively low, and patients had an increased number of infections. The absolute number of T, B, and NK cells were low but still in the normal range. Remarkably, all BS patients studied had a high percentage (20-80%) of CD4+ and CD8+ effector memory T cells. The process of SHM seems normal; however, the Ig subclass distribution was not normal, since the BS patients had more IGHG1 and IGHG3 transcripts. In conclusion, BS patients have low number of lymphocytes, but the immunodeficiency seems relatively mild since they have no severe or opportunistic infections. Most changes in the B cell development were seen in the CSR process; however, further studies are necessary to elucidate the exact role of BLM in CSR.
Project description:Bloom's syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1-BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.
Project description:Bloom's syndrome (BS) displays one of the strongest known correlations between chromosomal instability and a high risk of cancer at an early age. BS cells combine a reduced average fork velocity with constitutive endogenous replication stress. However, the response of BS cells to replication stress induced by hydroxyurea (HU), which strongly slows the progression of replication forks, remains unclear due to publication of conflicting results. Using two different cellular models of BS, we showed that BLM deficiency is not associated with sensitivity to HU, in terms of clonogenic survival, DSB generation, and SCE induction. We suggest that surviving BLM-deficient cells are selected on the basis of their ability to deal with an endogenous replication stress induced by replication fork slowing, resulting in insensitivity to HU-induced replication stress.
Project description:Bloom's syndrome (BS) is a rare human genetic disorder characterized by dwarfism, immunodeficiency, genomic instability and cancer predisposition. We have previously purified three complexes containing BLM, the helicase mutated in this disease. Here we demonstrate that BLAP75, a novel protein containing a putative OB-fold nucleic acid binding domain, is an integral component of BLM complexes, and is essential for their stability in vivo. Consistent with a role in BLM-mediated processes, BLAP75 colocalizes with BLM in subnuclear foci in response to DNA damage, and its depletion impairs the recruitment of BLM to these foci. Depletion of BLAP75 by siRNA also results in deficient phosphorylation of BLM during mitosis, as well as defective cell proliferation. Moreover, cells depleted of BLAP75 display an increased level of sister-chromatid exchange, similar to cells depleted of BLM by siRNA. Thus, BLAP75 is an essential component of the BLM-associated cellular machinery that maintains genome integrity.
Project description: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 ?-H2A at sites of Sgs1 binding, replication pausing regions, and long genes. The mutation signature of sgs1? 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.
Project description:Fanconi Anemia (FA) is an inherited multi-gene cancer predisposition syndrome that is characterized on the cellular level by a hypersensitivity to DNA interstrand crosslinks (ICLs). To repair these lesions, the FA pathway proteins are thought to act in a linear hierarchy: Following ICL detection, an upstream FA core complex monoubiquitinates the central FA pathway members FANCD2 and FANCI, followed by their recruitment to chromatin. Chromatin-bound monoubiquitinated FANCD2 and FANCI subsequently coordinate DNA repair factors including the downstream FA pathway members FANCJ and FANCD1/BRCA2 to repair the DNA ICL. Importantly, we recently showed that FANCD2 has additional independent roles: it binds chromatin and acts in concert with the BLM helicase complex to promote the restart of aphidicolin (APH)-stalled replication forks, while suppressing the firing of new replication origins. Here, we show that FANCD2 fulfills these roles independently of the FA core complex-mediated monoubiquitination step. Following APH treatment, nonubiquitinated FANCD2 accumulates on chromatin, recruits the BLM complex, and promotes robust replication fork recovery regardless of the absence or presence of a functional FA core complex. In contrast, the downstream FA pathway members FANCJ and BRCA2 share FANCD2's role in replication fork restart and the suppression of new origin firing. Our results support a non-linear FA pathway model at stalled replication forks, where the nonubiquitinated FANCD2 isoform - in concert with FANCJ and BRCA2 - fulfills a specific function in promoting efficient replication fork recovery independently of the FA core complex.