-
Yoh-ichi Kawabe,
Masayuki Seki,
Akari Yoshimura,
Katsuaki Nishino,
Tomoko Hayashi,
Takashi Takeuchi,
Sohta Iguchi,
Yumiko Kusa,
Makoto Ohtsuki,
Takashi Tsuyama, Osamu Imamura,
Takehisa Matsumoto,
Yasuhiro Furuichi,
Shusuke Tada,
Takemi Enomoto
[show abstract]
[hide abstract]
ABSTRACT: Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.
DNA Repair 08/2006; 5(7):816-28. · 4.14 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Yeast Dna2 helicase/nuclease is essential for DNA replication and assists FEN1 nuclease in processing a subset of Okazaki fragments that have long single-stranded 5' flaps. It is also involved in the maintenance of telomeres. DNA2 is a gene conserved in eukaryotes, and a putative human ortholog of yeast DNA2 (ScDNA2) has been identified. Little is known about the role of human DNA2 (hDNA2), although complementation experiments have shown that it can function in yeast to replace ScDNA2. We have now characterized the biochemical properties of hDna2. Recombinant hDna2 has single-stranded DNA-dependent ATPase and DNA helicase activity. It also has 5'-3' nuclease activity with preference for single-stranded 5' flaps adjacent to a duplex DNA region. The nuclease activity is stimulated by RPA and suppressed by steric hindrance at the 5' end. Moreover, hDna2 shows strong 3'-5' nuclease activity. This activity cleaves single-stranded DNA in a fork structure and, like the 5'-3' activity, is suppressed by steric hindrance at the 3'-end, suggesting that the 3'-5' nuclease requires a 3' single-stranded end for activation. These biochemical specificities are very similar to those of the ScDna2 protein, but suggest that the 3'-5' nuclease activity may be more important than previously thought.
Nucleic Acids Research 02/2006; 34(6):1865-75. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.
Proceedings of the National Academy of Sciences 08/2003; 100(14):8193-8. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have found that the Dna2 helicase-nuclease, thought to be involved in maturation of Okazaki fragments, is a component of telomeric chromatin. We demonstrate a dynamic localization of Dna2p to telomeres that suggests a dual role for Dna2p, one in telomere replication and another, unknown function, perhaps in telomere capping. Both chromatin immunoprecipitation (ChIP) and immunofluorescence show that Dna2p associates with telomeres but not bulk chromosomal DNA in G(1) phase, when there is no telomere replication and the telomere is transcriptionally silenced. In S phase, there is a dramatic redistribution of Dna2p from telomeres to sites throughout the replicating chromosomes. Dna2p is again localized to telomeres in late S, where it remains through G(2) and until the next S phase. Telomeric localization of Dna2p required Sir3p, since the amount of Dna2p found at telomeres by two different assays, one-hybrid and ChIP, is severely reduced in strains lacking Sir3p. The Dna2p is also distributed throughout the nucleus in cells growing in the presence of double-strand-break-inducing agents such as bleomycin. Finally, we show that Dna2p is functionally required for telomerase-dependent de novo telomere synthesis and also participates in telomere lengthening in mutants lacking telomerase.
Molecular and Cellular Biology 07/2002; 22(12):4202-17. · 5.53 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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. WRN and BLM genes causing WS and BS, encode proteins that are closely related to the RecQ helicase. We produced WRN-/-, BLM-/- and WRN(-/-)/BLM(-/-) mutants in the chicken B-cell line DT40. WRN-/- cells showed hypersensitivities to genotoxic agents, such as 4-nitroquinoline 1-oxide, camptothecin and methyl methanesulfonate. They also showed a threefold increase in targeted integration rate of exogenous DNAs, but not in sister chromatid exchange (SCE) frequency. BLM-/- cells showed hypersensitivities to the genotoxic agents as well as ultraviolet (UV) light, in addition to a 10-fold increase in targeted integration rate and an 11-fold increase in SCE frequency. In WRN(-/-)/BLM(-/-) cells, synergistically increased hypersensitivities to the genotoxic agents were observed whereas both SCE frequencies and targeted integration rates were partially diminished compared to the single mutants. Chromosomal aberrations were also synergistically increased in WRN(-/-)/BLM(-/-) cells when irradiated with UV light in late S to G(2) phases. These results suggest that both WRN and BLM may be involved in DNA repair in a complementary fashion.
Oncogene 02/2002; 21(6):954-63. · 6.37 Impact Factor
-
Takehisa Matsumoto, Osamu Imamura,
Yukako Yamabe,
Junro Kuromitsu,
Yoshiki Tokutake,
Akira Shimamoto,
Noriyuki Suzuki,
Misako Satoh,
Saori Kitao,
Koji Ichikawa,
Hiroshi Kataoka,
Kahori Sugawara,
Winston Thomas,
Brian Mason,
Zenta Tsuchihashi,
D. Drayna,
Minoru Sugawara,
Masanobu Sugimoto,
Y. Furuichi,
Makoto Goto
[show abstract]
[hide abstract]
ABSTRACT: The correlation between mutations in the Werner’s syndrome (WRN) gene and the haplotypes of surrounding markers was studied
in Japanese patients. We have elucidated the genomic structure of WRN helicase, and found five additional mutations, designated
mutations 6–10. Mutations 4 and 6 were found to be the two major mutations in this population; these mutations comprised 50.8%
and 17.5%, respectively, of the total in a sample of 126 apparently unrelated chromosomes. Almost all the patients homozygous
for mutation 4 shared a haplotype around the WRN gene, consistent with the view that they are derived from a single ancestor.
This important advantage demonstrated in the identification of the WRN gene suggests that the Japanese present a unique population
for the cloning of other disease genes. The conserved haplotype was observed across 19 loci, extending a distance estimated
to be more than 1.4 Mbp around the WRN gene. This haplotype is rare among random Japanese individuals. Unexpectedly, all the
nine patients homozygous for mutation 6 shared a haplotype that was identical to this haplotype at 18 of these 19 markers.
These results suggest that mutations 4 and 6 arose independently in almost identical rare haplotypes. The remaining mutations
(1, 5, 7, 8, 9, and 10) occurred rarely, and were each associated with different haplotypes.
Human Genetics 05/1997; 100(1):123-130. · 5.07 Impact Factor
-
Makaoto Goto, Osamu Imamura,
Junro Kuromitsu,
Takehisa Matsumoto,
Yukako Yamabe,
Yoshiki Tokutake,
Noriyuki Suzuki,
Brian Mason,
Dennis Drayna,
Minoru Sugawara,
Masanobu Sugimoto,
Y. Furuichi
[show abstract]
[hide abstract]
ABSTRACT: The profile of helicase gene mutations was studied in 89 Japanese Werner’s syndrome (WRN) patients by examining the previously
described mutations 1– 4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89
patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained
mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none
of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes,
including those carrying mutations of both types 1 and 4. The genotype analysis of the markers surrounding the WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single
founders.
Human Genetics 12/1996; 99(2):191-193. · 5.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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. Among the six up-regulated mRNAs were three mRNAs that coded TGFβ-1 and two proteins, their expressions of which were increased by TGFβ-1. These results together with the fact that TGFβ-1 up-regulates the expression of ECM proteins strongly suggest that TGFβ-1 has a key role in accelerated cellular senescence of fibroblasts of WS patients.
Mechanisms of Ageing and Development. 115(3):189-198.
-
Koji Ichikawa,
Akira Shimamoto, Osamu Imamura,
Yoshiki Tokutake,
Yukako Yamabe,
Saori Kitao,
Noriyuki Suzuki,
Kahori Sugawara,
Takehisa Matsumoto,
Winston Thomas,
Dennis Drayna,
Makoto Goto,
Masanobu Sugimoto,
Minoru Sugawara,
Yasuhiro Furuichi
[show abstract]
[hide abstract]
ABSTRACT: 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. The map comprises a series of contigs mostly P1/PAC clones, which span the loci of potential tumor suppressor genes and the Werner's syndrome gene. Each P1/PAC DNA was defined by its size, restriction sites, terminal sequences, intermarker distances and location relative to major genes and markers. The genes on these P1/PAC DNAs were analyzed by an exon amplification method to determine their locations. The genes newly found by the exon amplification method together with other known genes, including those of glutathion reductase, a general transcription factor, protein phosphatase 2A β subunit and Werner's syndrome, were precisely mapped within the contigs. These P1/PAC DNAs are useful reagents for the generation of new microsatellite markers to narrow the candidate region of the tumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in this region by linkage analysis.
-
Koji Ichikawa,
Yukako Yamabe, Osamu Imamura,
Junro Kuromitsu,
Kahori Sugawara,
Noriyuki Suzuki,
Akira Shimamoto,
Takehisa Matsumoto,
Yoshiki Tokutake,
Saori Kitao,
Hiroshi Kataoka,
Misako Satoh,
Masanobu Sugimoto,
Makoto Goto,
Minoru Sugawara,
Yasuhiro Furuichi
[show abstract]
[hide abstract]
ABSTRACT: A novel human gene referred to as the WS-3 gene, in the short arm of human chromosome 8, was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR (TAIL-PCR) and the Marathon-Ready cDNA amplification method. The gene consists of 7 exons separated by 6 introns, and is at the telomere side of the STS marker, D8S1055. The full-length WS-3 gene contains 1052 nucleotides and codes for a protein of 190 amino acids with a calculated mol. wt. of 20 747. Southern blot experiments showed that the WS-3 gene exists as a single copy in the human genome. A protein encoded by the WS-3 gene has an R-G-D (Arg-Gly-Asp) motif in the N-terminal region, which seems to confer adhesive properties to macromolecular proteins like fibronectin. Although WS-3 is a small gene with unknown biological function, its ubiquitous expression in various tissues and organs suggests that the encoded protein is one of the essential components of all organs and tissues.
Gene.
