RECQ DNA Helicases and Osteosarcoma.

The RECQ family of DNA helicases is a conserved group of enzymes that plays an important role in maintaining genomic stability. Humans possess five RECQ helicase genes, and mutations in three of them - BLM, WRN, and RECQL4 - are associated with the genetic disorders Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome (RTS), respectively. These syndromes share overlapping clinical features, and importantly they are all associated with an increased risk of cancer. Patients with RTS have the highest specific risk of developing osteosarcoma compared to all other cancer predisposition syndromes; therefore, RTS serves as a relevant model to study the pathogenesis and molecular genetics of osteosarcoma. The "tumor suppressor" function of the RECQ helicases continues to be an area of active investigation. This chapter will focus primarily on the known cellular functions of RECQL4 and how these may relate to tumorigenesis, as well as ongoing efforts to understand RECQL4's functions in vivo using animal models. Understanding the RECQ pathways will provide insight into avenues for novel cancer therapies in the future.

Chromosome alignment maintenance requires the MAP RECQL4, mutated in the Rothmund-Thomson syndrome.

RecQ-like helicase 4 (RECQL4) is mutated in patients suffering from the Rothmund-Thomson syndrome, a genetic disease characterized by premature aging, skeletal malformations, and high cancer susceptibility. Known roles of RECQL4 in DNA replication and repair provide a possible explanation of chromosome instability observed in patient cells. Here, we demonstrate that RECQL4 is a microtubule-associated protein (MAP) localizing to the mitotic spindle. RECQL4 depletion in M-phase-arrested frog egg extracts does not affect spindle assembly per se, but interferes with maintaining chromosome alignment at the metaphase plate. Low doses of nocodazole depolymerize RECQL4-depleted spindles more easily, suggesting abnormal microtubule-kinetochore interaction. Surprisingly, inter-kinetochore distance of sister chromatids is larger in depleted extracts and patient fibroblasts. Consistent with a role to maintain stable chromosome alignment, RECQL4 down-regulation in HeLa cells causes chromosome misalignment and delays mitotic progression. Importantly, these chromosome alignment defects are independent from RECQL4's reported roles in DNA replication and damage repair. Our data elucidate a novel function of RECQL4 in mitosis, and defects in mitotic chromosome alignment might be a contributing factor for the Rothmund-Thomson syndrome.

Human RecQL4 helicase plays multifaceted roles in the genomic stability of normal and cancer cells.

Human RecQ helicases that share homology with E. coli RecQ helicase play critical roles in diverse biological activities such as DNA replication, transcription, recombination and repair. Mutations in three of the five human RecQ helicases (RecQ1, WRN, BLM, RecQL4 and RecQ5) result in autosomal recessive syndromes characterized by accelerated aging symptoms and cancer incidence. Mutational inactivation of Werner (WRN) and Bloom (BLM) genes results in Werner syndrome (WS) and Bloom syndrome (BS) respectively. However, mutations in RecQL4 result in three human disorders: (I) Rothmund-Thomson syndrome (RTS), (II) RAPADILINO and (III) Baller-Gerold syndrome (BGS). Cells from WS, BS and RTS are characterized by a unique chromosomal anomaly indicating that each of the RecQ helicases performs specialized function(s) in a non-redundant manner. Elucidating the biological functions of RecQ helicases will enable us to understand not only the aging process but also to determine the cause for age-associated human diseases. Recent biochemical and molecular studies have given new insights into the multifaceted roles of RecQL4 that range from genomic stability to carcinogenesis and beyond. This review summarizes some of the existing and emerging knowledge on diverse biological functions of RecQL4 and its significance as a potential molecular target for cancer therapy.

Understanding photodermatoses associated with defective DNA repair: Syndromes with cancer predisposition.

Hereditary photodermatoses are a spectrum of rare photosensitive disorders that are often caused by genetic deficiency or malfunction of various components of the DNA repair pathway. This results clinically in extreme photosensitivity, with many syndromes exhibiting an increased risk of cutaneous malignancies. This review will focus specifically on the syndromes with malignant potential, including xeroderma pigmentosum, Bloom syndrome, and Rothmund-Thomson syndrome. The typical phenotypic findings of each disorder will be examined and contrasted, including noncutaneous identifiers to aid in diagnosis. The management of these patients will also be discussed. At this time, the mainstay of therapy remains strict photoprotection; however, genetic therapies are under investigation.

The Rothmund-Thomson syndrome helicase RECQL4 is essential for hematopoiesis.

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.

Senescence induced by RECQL4 dysfunction contributes to Rothmund-Thomson syndrome features in mice.

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 ß-galactosidase (SA-ß-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-ß-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.

C16orf57, a gene mutated in poikiloderma with neutropenia, encodes a putative phosphodiesterase responsible for the U6 snRNA 3' end modification.

C16orf57 encodes a human protein of unknown function, and mutations in the gene occur in poikiloderma with neutropenia (PN), which is a rare, autosomal recessive disease. Interestingly, mutations in C16orf57 were also observed among patients diagnosed with Rothmund-Thomson syndrome (RTS) and dyskeratosis congenita (DC), which are caused by mutations in genes involved in DNA repair and telomere maintenance. A genetic screen in Saccharomyces cerevisiae revealed that the yeast ortholog of C16orf57, USB1 (YLR132C), is essential for U6 small nuclear RNA (snRNA) biogenesis and cell viability. Usb1 depletion destabilized U6 snRNA, leading to splicing defects and cell growth defects, which was suppressed by the presence of multiple copies of the U6 snRNA gene SNR6. Moreover, Usb1 is essential for the generation of a unique feature of U6 snRNA; namely, the 3'-terminal phosphate. RNAi experiments in human cells followed by biochemical and functional analyses confirmed that, similar to yeast, C16orf57 encodes a protein involved in the 2',3'-cyclic phosphate formation at the 3' end of U6 snRNA. Advanced bioinformatics predicted that C16orf57 encodes a phosphodiesterase whose putative catalytic activity is essential for its function in vivo. Our results predict an unexpected molecular basis for PN, DC, and RTS and provide insight into U6 snRNA 3' end formation.

RECQL4 is essential for the transport of p53 to mitochondria in normal human cells in the absence of exogenous stress.

Mutations in RECQL4 helicase are associated with Rothmund-Thomson syndrome (RTS). A subset of RTS patients is predisposed to cancer and is sensitive to DNA damaging agents. The enhanced sensitivity of cells from RTS patients correlates with the accumulation of transcriptionally active nuclear p53. We found that in untreated normal human cells these two nuclear proteins, p53 and RECQL4, instead colocalize in the mitochondrial nucleoids. RECQL4 accumulates in mitochondria in all phases of the cell cycle except S phase and physically interacts with p53 only in the absence of DNA damage. p53-RECQL4 binding leads to the masking of the nuclear localization signal of p53. The N-terminal 84 amino acids of RECQL4 contain a mitochondrial localization signal, which causes the localization of RECQL4-p53 complex to the mitochondria. RECQL4-p53 interaction is disrupted after stress, allowing p53 translocation to the nucleus. In untreated normal cells RECQL4 optimizes de novo replication of mtDNA, which is consequently decreased in fibroblasts from RTS patients. Wild-type RECQL4-complemented RTS cells show relocalization of both RECQL4 and p53 to the mitochondria, loss of p53 activation, restoration of de novo mtDNA replication and resistance to different types of DNA damage. In cells expressing Δ84 RECQL4, which cannot translocate to mitochondria, all the above functions are compromised. The recruitment of p53 to the sites of de novo mtDNA replication is also regulated by RECQL4. Thus these findings elucidate the mechanism by which p53 is regulated by RECQL4 in unstressed normal cells and also delineates the mitochondrial functions of the helicase.

Rothmund-Thomson syndrome helicase, RECQ4: on the crossroad between DNA replication and repair.

RECQ proteins are conserved DNA helicases in both prokaryotes and eukaryotes. The importance of the RECQ family helicases in human health is demonstrated by their roles as cancer suppressors that are vital for preserving genome integrity. Mutations in one of the RECQ family proteins, RECQ4, not only result in developmental abnormalities and cancer predispositions, but are also linked to premature aging. Therefore, defining the function and regulation of the RECQ4 protein is fundamental to our understanding of both the aging process and cancer pathogenesis. This review will summarize the clinical effect of RECQ4 in human health, and discuss the recent progress and debate in defining the complex molecular function of RECQ4 in DNA metabolism.

The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability.

Rothmund-Thomson syndrome (RTS) is a human genetic disorder characterized by genome instability, cancer susceptibility and premature aging. The gene defective in a subset of RTS cases, RECQL4, encodes a member of the RecQ family of DNA helicases. To better define the function of the RECQL4 protein, we have determined its subcellular localization. We have raised antibodies against the N- and C-terminal parts of RECQL4 and could show that in various human cells endogenous RECQL4 forms discrete nuclear foci that colocalize with promyelotic leukaemia protein (PML). The number of foci and their colocalization with PML does not significantly change after induction of different types of DNA damages. Silencing of RECQL4 expression by siRNA causes a significant reduction in RECQL4 nuclear foci formation. Furthermore, we demonstrate that RECQL4 foci coincide with foci formed by human Rad51 and regions of single-stranded DNA after induction of DNA double-strand breaks. In agreement with this, we also show that RECQL4 and Rad51 form a complex in human cells. Our findings suggest a role for RECQL4 in the repair of DNA double-strand breaks by homologous recombination and shed new light onto RECQL4's function in human cells.

Initiation of DNA replication requires the RECQL4 protein mutated in Rothmund-Thomson syndrome.

How the replication machinery is loaded at origins of DNA replication is poorly understood. Here, we implicate in this process the Xenopus laevis homolog (xRTS) of the RECQL4 helicase mutated in Rothmund-Thomson syndrome. xRTS, which bears homology to the yeast replication factors Sld2/DRC1, is essential for DNA replication in egg extracts. xRTS can be replaced in extracts by its human homolog, while RECQL4 depletion from mammalian cells induces proliferation failure, suggesting an evolutionarily conserved function. xRTS accumulates on chromatin during replication initiation, after prereplication-complex (pre-RC) proteins, Cut5, Sld5, or Cdc45 but before replicative polymerases. xRTS depletion suppresses the loading of RPA, the ssDNA binding protein that marks unwound origins before polymerase recruitment. However, xRTS is unaffected by xRPA depletion. Thus, xRTS functions after pre-RC formation to promote loading of replication factors at origins, a previously unrecognized activity necessary for initiation. This role connects defective replication initiation to a chromosome-fragility disorder.

RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund-Thomson patient.

Rothmund-Thomson syndrome (RTS) (OMIM 268400) is an autosomal recessive genodermatosis associated with genomic instability and increased risk of mesenchymal cancers. Mutations in the RECQL4 gene, encoding a protein of the family of Werner (WRN) and Bloom (BLM) helicases, have been identified in a subset of RTS patients. Apart from congenital poikiloderma, the clinical presentation of RTS is widely variable, raising the question of the possible existence of a second locus. Results herein reported on a sporadic Caucasian patient emphasize the concept that mutation analyses at both DNA and RNA level complement the genetic defect suggested by clinical and cytogenetic signs. The patient presented with typical congenital poikiloderma and bone defects and exhibited significant genomic instability in the peripheral blood karyotype. By RECQL4 DNA mutation analysis, he was found to carry a 1473delT (mut 5) on one allele and an AG to AC change at the 3'-splice site of exon 13 (a variant of mut 4) on the second allele. RT-PCR analysis of RECQL4 cDNA encompassing the entire helicase domain showed diffuse splicing defects indicating that the loss of a single 3'-splice signal motif disregulates the correct splice-site selection and affects the overall RNA processing. The presence of an unstable minisatellite which ends at 3'-splice site of IVS12 may enhance the mutation at this site. This genomic feature together with a number of short introns in the RECQL4 gene may account for the common missplicing of RECQL4 mRNA. While it is possible that defects of RECQL4 mRNA processing might account for part of the clinical variability observed for this syndrome, only a thorough analysis at both genomic and RNA level may allow a genotype-phenotype correlation in RTS patients, restricting the search of a second RTS locus to the specific patients.

Association between osteosarcoma and deleterious mutations in the RECQL4 gene in Rothmund-Thomson syndrome.

BACKGROUND: Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder associated with an increased predisposition to osteosarcoma. Children with RTS typically present with a characteristic skin rash (poikiloderma), small stature, and skeletal dysplasias. Mutations in the RECQL4 gene, which encodes a RecQ DNA helicase, have been reported in a few RTS patients. We examined whether a predisposition to developing osteosarcoma among an international cohort of RTS patients was associated with a distinctive pattern of mutations in the RECQL4 gene. METHODS: We obtained clinical information about and biologic samples from 33 RTS patients (age range = 1-30 years). Eleven patients were diagnosed with osteosarcoma. All 21 exons and 13 short introns of the RECQL4 gene were sequenced from the genomic DNA of all subjects. Kaplan-Meier survival analysis was used to estimate the incidence of osteosarcoma among patients with and without mutations predicted to produce a truncated RECQL4 protein. RESULTS: Twenty-three RTS patients, including all 11 osteosarcoma patients, carried at least one of 19 truncating mutations in their RECQL4 genes. The incidence of osteosarcoma was 0.00 per year in truncating mutation-negative patients (100 person-years of observation) and 0.05 per year in truncating mutation-positive patients (230 person-years of observation) (P =.037; two-sided log-rank test). CONCLUSIONS: Mutations predicted to result in the loss of RECQL4 protein function occurred in approximately two-thirds of RTS patients and are associated with risk of osteosarcoma. Molecular diagnosis has the potential to identify those children with RTS who are at high risk of this cancer.

RecQ helicases: caretakers of the genome.

RecQ helicases are highly conserved from bacteria to man. Germline mutations in three of the five known family members in humans give rise to debilitating disorders that are characterized by, amongst other things, a predisposition to the development of cancer. One of these disorders--Bloom's syndrome--is uniquely associated with a predisposition to cancers of all types. So how do RecQ helicases protect against cancer? They seem to maintain genomic stability by functioning at the interface between DNA replication and DNA repair.

Intron-size constraint as a mutational mechanism in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder caused by deleterious mutations in the RECQL4 gene on chromosome 8. The RECQL4 gene structure is unusual because it contains many small introns <100 bp. We describe a proband with RTS who has a novel 11-bp intronic deletion, and we show that this mutation results in a 66-bp intron too small for proper splicing. Constraint on intron size may represent a general mutational mechanism, since human-genome analysis reveals that approximately 15% of genes have introns <100 bp and are therefore susceptible to size constraint. Thus, monitoring of intron size may allow detection of mutations missed by exon-by-exon approaches.

Premature aging and predisposition to cancers caused by mutations in RecQ family helicases.

DNA helicases, because they unwind duplex DNA, have important roles in cellular DNA events such as replication, recombination, repair, and transcription. Multiple DNA helicase families with seven consensus motifs have been found, and members within each helicase family also share sequence homologies between motifs. The RecQ helicase family includes helicases that have extensive amino acid sequence homologies to the E. coli DNA helicase RecQ, which has been implicated in double-strand break repair and suppression of illegitimate recombination. To date, five RecQ helicase species exist in humans, but their exact biological functions remain unknown. In this paper, on the basis of five years of work, I overview the updated molecular biology of five human RecQ helicases; genetic diseases such as Werner's, Bloom's, and Rothmund-Thomson's syndromes caused by helicase mutations; the associated premature aging phenotype; and an increased risk of neoplasms. I also describe a hypothesis of "tissue-specific genomic instability" that accounts for the pathology behind multisymptomatic RecQ helicase syndromes.