2001 Fiscal Year Final Research Report Summary
Analysis for spatiotemporal regulation of DNA replication intiation proteins on human globin loci.
| Project/Area Number |
12680683
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Aichi Cancer Center |
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Principal Investigator |
FUJITA Masatoshi Aichi Cancer Center, Laboratory of Viral Oncology, Senior Researcher, 腫瘍ウイルス学部, 主任研究員 (30270713)
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| Project Period (FY) |
2000 – 2001
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| Keywords | Human cells / Regulation for DNA replication initiation / Cell cycle / ORC protein / CDC6 protein / MCM protein / Globin locujs / Chromatin immunoprecipitation |
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| Research Abstract |
The purpose of this project is to understand spatiotemporal regulation of DNA replication initiation proteins such as ORC, CDC6 and MCM in mammalian cells. We have investigated nuclear organization and cell cycle control of these proteins. We found that ORC1 and ORC2 proteins are associated with the nuclear matrix and physically interact with each other. Therefore, it is likely that ORC1 forms a complex with ORC2, 3, 4 and 5 on the nuclear matrix, probably functioning in DNA replication. ORC1 proteins were found to be degraded during S phase by an ubiquitin-proteasome dependent pathway. This may contribute to bind to the nuclear matrix although their physical association with ORC 1 has been undetectable. On the other hand, our results suggest that most MCM complexes are more widely distributed on chromatin beyond ORC foci. Such organization could provide an explanation for "initiation zone of DNA replication" suggested in mammalian cells. By chromatin immunoprecipitation assay, we have tried to determine the binding pattern of these proteins to chromosomal DNA of human globin locus, which has been suggested to act as a DNA replication unit. We first established semi-quantitative PCR assay to detect seven representative fragments on the locus, containing a putative replication origin upstream of beta-globin gene. Our current preliminary results with asynchronous HeLa cells suggest that MCM complexes may be associated with all regions tested, with some preferential binding to the origin. We do not yet obtain indicative results regarding CDC6 and ORC binding pattern. We continue trying to obtain the conclusive data with chromatin immunoprecipitation assay.
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[Publications] Fujii, K., Yokoyama, N., Kiyono, T., Kuzushima, K., Homma, M., Nishiyama, Y., Fujita, M., and Tsurumi, T.: "The Epstein-Barr virus pol catalytic subunit physically interacts with the BBLF4/BSLF1/BBLF2/3 complex"J. Virol.. 74. 2550-2557 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Yokoyama, N., Hirata, M., Ohtsuka, K., Fujii, K., Fujita, M., Kuzushima, K., Kiyono, T. and Tsurumi, T.: "Co-expression of human chaperone Hsp70 and Hsdj or Hsp40 co-factor increases solubility of overexpressed target proteins"Biochim. Biophys. Acta. 1493. 119-124 (2000)
Description
「研究成果報告書概要(欧文)」より
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