Quantitative analysis of Werner helicase activity using the single-molecule fluorescence detection system MF10S.

We developed a system that uses the single-molecule fluorescence detection system MF10S to assess quantitatively the activity of WRN helicase, the product of the causative gene of Werner syndrome that includes premature ageing. Double-strand DNA substrates labeled with the fluorescence dye TAMRA at the 5' end and with a quencher at the 3' end of the counter strand were incubated with a single trapper oligonucleotide and Werner helicase, and the resultant single DNA fragments labeled with TAMRA produced by the unwinding of WRN helicase were detected using the MF10S. The results using this system and those using polyacrylamide gel electrophoresis were well correlated.

Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus: modulation by cellular immortalization and senescence.

B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus have a phenotype corresponding to activated B-lymphoblasts. Although they are widely used as models in various biological and medical studies, their innate morphological differentiation and apoptosis has been little studied. We report here that a large proportion of LCL cells spontaneously differentiate into smaller lymphoid cells which ultimately undergo apoptosis during conventional cell culture.

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex.

Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging. The gene (WRN) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a nucleolar localization signal and shows intranuclear trafficking between the nucleolus and the nucleoplasm.

Werner and Bloom helicases are involved in DNA repair in a complementary fashion.

Werner syndrome (WS) is a recessive disorder characterized by premature senescence. Bloom syndrome (BS) is a recessive disorder characterized by short stature and immunodeficiency. A common characteristic of both syndromes is genomic instability leading to tumorigenesis.

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.

Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51.

Background: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family.

Diverged nuclear localization of Werner helicase in human and mouse cells.

Werner syndrome (WS) is a rare autosomal recessive genetic disorder causing premature aging and rare cancers. A gene responsible for WS (WRN) encodes a protein with 1432 amino acids (a.a.

Bloom helicase is involved in DNA surveillance in early S phase in vertebrate cells.

Bloom syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and an elevated risk of malignancy. The gene mutated in BS, BLM, encodes a RecQ-type DNA helicase. BS cells have mutator phenotypes such as hyper-recombination, chromosome instability and an increased frequency of sister chromatid exchange (SCE).

Viral and cellular mRNA capping: past and prospects.

This chapter focuses on the history of the discovery of cap and an update of research on viral and cellular-messenger RNA (mRNA) capping. Cap structures of the type m GpppN(m)pN(m)p are present at the 5′ ends of nearly all eukaryotic cellular and viral mRNAs. A cap is added to cellular mRNA precursors and to the transcripts of viruses that replicate in the nucleus during the initial phases of transcription and before other processing events, including internal NA methylation, 3′-poly (A) addition, and exon splicing.

Differential regulation of human RecQ family helicases in cell transformation and cell cycle.

Three human RecQ DNA helicases, WRN, BLM and RTS, are involved in the genetic disorders associated with genomic instability and a high incidence of cancer. RecQL1 and RecQL5 also belong to the human RecQ helicase family, but their correlation with genetic disorders, if any, is unknown. We report here that in human B cells transformed by Epstein-Barr virus (EBV), human fibroblasts and umbilical endothelial cells transformed by simian virus 40, the expression of WRN, BLM, RTS and RecQL1 was sharply up-regulated.

Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A.

We describe the unique structural features of a large telomere repeat DNA complex (TRDC) of >20 kb generated by a simple PCR using (TTAGGG)(4) and (CCCTAA)(4) as both primers and templates. Although large, as determined by conventional agarose gel electrophoresis, the TRDC was found to consist of short single-stranded DNA telomere repeat units of between several hundred and 3000 bases, indicating that it is a non-covalent complex comprising short cohesive telomere repeat units. S1 nuclease digestion showed that the TRDC contains both single- and double-stranded portions stable enough to survive glycerol density gradient centrifugation, precipitation with ethanol and gel electrophoresis.

Overexpression of mRNAs of TGFbeta-1 and related genes in fibroblasts of Werner syndrome patients.

We analyzed mRNAs that were up- or down-regulated in fibroblasts from Werner syndrome (WS) patients compared with those from normal individuals. The mRNAs from normal and WS cells were first screened by differential display, and those mRNAs that were apparently up- or down-regulated were selected except for mRNAs related to extra-cellular matrix (ECM) proteins that are already known to be up-regulated in WS fibroblasts. Then, the expression levels of these mRNAs were semiquantified by northern blot analysis, and six up-regulated and two down-regulated mRNAs were identified in WS cell lines.

Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA.

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge.

Human RecQ5beta, a large isomer of RecQ5 DNA helicase, localizes in the nucleoplasm and interacts with topoisomerases 3alpha and 3beta.

The RecQ helicase superfamily has been implicated in DNA repair and recombination. At least five human RecQ-related genes exist: RecQ1, BLM, WRN, RecQ4 and RecQ5. Mutations in BLM, WRN and RecQ4 are associated with Bloom, Werner and Rothmund-Thomson syndromes, respectively, involving a predisposition to malignancies and a cellular phenotype that includes increased chromosome instability.

Rothmund-thomson syndrome responsible gene, RECQL4: genomic structure and products.

RECQL4 is the fourth gene identified as a member of the human DNA helicase RecQ gene family including the genes for Werner syndrome (WRN) and Bloom syndrome, both of which are characterized by genomic instability. Recently, RECQL4 was identified as the gene responsible for some cases of Rothmund-Thomson syndrome (RTS), a rare autosomal recessive genetic disorder that shows chromosomal instability, premature aging, and a high risk of mesenchymal tumors. In this study, we show the genomic organization of the RECQL4 gene, including the exon-intron boundaries, the transcription initiation sites, and the potential promoter sequences, which facilitates further mutation analysis of the RECQL4 gene and studies to elucidate the pathogenesis behind RTS.

Werner syndrome helicase contains a 5'-->3' exonuclease activity that digests DNA and RNA strands in DNA/DNA and RNA/DNA duplexes dependent on unwinding.

We show that WRN helicase contains a unique 5'-->3' exonuclease activity in the N-terminal region. Adeletion mutant lacking 231 N-terminal amino acid residues, made in a baculovirus system, did nothave this activity, while it showed ATPase and DNA helicase activities. This exonuclease activity was co-precipitated with the helicase activity using monoclonal antibodies specific to WRN helicase, indicating that it is an integral component with WRN helicase.

Mutations in RECQL4 cause a subset of cases of Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS; also known as poikiloderma congenitale) is a rare, autosomal recessive genetic disorder characterized by abnormalities in skin and skeleton, juvenile cataracts, premature ageing and a predisposition to neoplasia. Cytogenetic studies indicate that cells from affected patients show genomic instability often associated with chromosomal rearrangements causing an acquired somatic mosaicism. The gene(s) responsible for RTS remains unknown.

Reconsideration of senescence, immortalization and telomere maintenance of Epstein-Barr virus-transformed human B-lymphoblastoid cell lines.

We review recent data on senescence and immortalization of human B-lymphoblastoid cell lines (LCLs) transformed by the Epstein-Barr virus (EBV). Although EBV-transformed LCLs are generally believed to be immortalized, a series of recent studies, including ours, provided strong evidence that they are mostly mortal and have non-malignant properties, except for a small proportion of LCLs that are immortalized by developing a strong telomerase activity. A large proportion of mortal LCLs have exceptionally long lifespans.

Structures of mouse Rep-8 cDNA and genomic clones.

A mouse homologue of the human Rep-8 gene was cloned by PCR methods using degenerate oligonucleotide primers corresponding to highly conserved regions between human and mouse genes, and by the Marathon-Ready cDNA amplification method. The full-length mouse Rep-8 contains 1422 nucleotides and codes for a protein of 277 amino acids with a calculated mol. wt.

Detection by epitope-defined monoclonal antibodies of Werner DNA helicases in the nucleoplasm and their upregulation by cell transformation and immortalization.

We prepared several monoclonal antibodies (mAbs) specific for the NH2- and COOH-terminal regions of the DNA helicase (WRN helicase) responsible for Werner's syndrome known as a premature aging disease. With these antibodies, we detected by immunoblot analysis the endogenous WRN helicase of a relative mass of 180 kD in several lines of cultured cells, but not in patient cells with a defined mutation. Immunocytochemical staining of proliferating fibroblasts and tumor cells showed that the major part of WRN helicase is in the nucleoplasm and not in the nucleolus.

Cloning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes.

Two new human DNA helicase genes, RecQ4 and RecQ5, that belong to the RecQ helicase family were cloned and characterized. The addition of these genes increases the total to five helicase genes in the human RecQ family, which includes helicases involved in Bloom and Werner syndromes, the genetic diseases manifesting the distinctive but overlapping clinical phenotypes of immunodeficiency, premature aging, and an enhanced risk of cancer. The RecQ4 helicase is as large as the Bloom (BLM) and Werner (WRN) helicases, and its gene expression profile is organ-specific, resembling that of BLM helicase.

Characterization of the nuclear localization signal in the DNA helicase involved in Werner's syndrome.

The nuclear localization signal (NLS) of the DNA helicase involved in Werner's syndrome (WS) was studied. Previously, we noted that the C-terminal region of WS helicase contains the NLS. In this study, we generated in HeLa cells various chimeric proteins consisting of the N-terminal tagged with an enhanced green fluorescent protein and the C-terminal fragments of the WS helicase that were truncated either from N- or C-termini, and we examined the ability of fragments to transfer the fusion proteins to the nucleoplasm by fluorescence microscopy.

Cloning and characterization of human Sep1 (hSEP1) gene and cytoplasmic localization of its product.

We isolated and sequenced a human cDNA (designated as hSEP1) encoding both a homologue of mouse Dhm2 and budding yeast SEP1. The gene was shown to be located on the long arm of chromosome 3 (3q25-26.1).

Sp1-mediated transcription of the Werner helicase gene is modulated by Rb and p53.

The regulation of Werner's syndrome gene (WRN) expression was studied by characterizing the cis-regulatory elements in the promoter region and the trans-activating factors that bind to them. First, we defined the transcription initiation sites and the sequence of the 5' upstream region (2.8 kb) of WRN that contains a number of cis-regulatory elements, including 7 Sp1, 9 retinoblastoma control element (RCE), and 14 AP2 motifs.

Physical map of the human chromosome 8p12-p21 encompassing tumor suppressor and Werner's syndrome gene loci.

Detailed physical maps of the human genome are important resources for identification and isolation of genes responsible for diseases and for the study of their structure and function. We constructed a 2.0-Mb high-resolution physical map within the human chromosome 8p12-p21 region extending from marker D8S131 to D8S283.