| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Werner syndrome (WS) is a potential model of accelerated human aging. The gene responsible WS encodes a protein homologous to Escherichia coli recQ. We identified three proteins interacting with Werner protein (WRN). One was a novel Werner helicase interacting protein (WHIP), which shows homology to replication factor C family proteins. The other two were UBC9 and SUMO. UBC9 also interacted with Bloom syndrome gene product (BLM), another eukaryotic RecQ-type helicase. UBC9 is involved in SUMO conjugation of various target proteins. Supported by this grant, we have made several important findings as follow.
1) We demonstrated a covalent association of WRN with SUMO in cells.
2) Exogenously expressed BLM formed dots/rod-like structures which are associated with SUMO in the nuclei.
3) Disruption of the yeast WHIP gene in wild-type cells and sgs1 (WRN homologue) disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotypes of sgs1, respectively. In contr
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ast, disruption of the WHIP gene partially alleviated the sensitivity to methylmethanesulfonate of sgs1.
4) Disruption of chicken WHIP gene in chicken B-lymphocyte DT40 wild-type cells and BLM-/- cells resulted in no phenotypes and the exaggerated phenotypes of DT40 BLM-/- cells, respectively, suggesting that higher vertebrate WHIP is involved in the maintenance of genome integrity at least in the absence of BLM.
5) Yeast Whip overexpression causes lethality in combination with mutations in replication proteins such as Polδ, RFC, PCNA, and RPA. Genetic analysis of double mutants confirmed that Whip interacts functionally with Polδ and RFC.
6) Both Sgs1 and BLM interacted with Top3, functionally and physically. We demonstrated here that Top3 is involved in DNA recombination repair. In addition, yeast Ubc9 is required for proper regulation of DNA recombination repair.
Taken all together, eukaryotic RecQ helicases and interacting protein with RecQs may be involved in the regulation of homologous recombination coupled with DNA replication. Further study based on this achievement, will shed light the molecular mechanisms of aging. Less
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