1991 Fiscal Year Final Research Report Summary
Molecular Biological Analysis of the Effect of UV Irradiation and Repair Mechanism of UV Damaged DNA.
| Project/Area Number |
02671026
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
MURAKAMI Yasufumi Institute of Chemical And Physical Reseach, Department of Genebank Research Scientist, ライフサイエンス筑波研究センター・ジーンバンク室, 研究員 (90200279)
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| Project Period (FY) |
1990 – 1991
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| Keywords | UV irradiation / DAN damage / DNA repair / DNA replication / pyrimidine dimer / ozone / SV40 / replication mechanism |
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| Research Abstract |
In order to analyze molecular mechanism how UV irradiation inhibits DNA replication and to analyze molecular mechanism of repair opf UV damaged DNA, SV40 DNA replication system in vitro has been chosen as a model system. SV40 replication origin containing DNA was UV irradiated and the replication activity has been assayed. In the presence of T antigen, replication reaction occurres efficiently in this system. UV irradiated DNA showed little replication activity in the presence of T antigen. In the absence of T antigen, UV irradiation DNA synthesis has been observed in a UV dose dependent manner. To have maximum incorporation, the conditions for DNA synthesis has been optimized. Under optimized condition, picomole order of dNTP incorporation has been observed while non-specific DNA synthesis occurred in a very low level.
To determine the amount of pyrimidine dimer, a simple assay system has been developed_using restriction enzymes and agarose gel electrophoresis. By this assay, it has been suggested that removal of the pyrimidine dimer has taken place through incubation carried out in the optimum condition.
The effect of UV irradiation on DNA replication reaction has been analyzed using SV40 DNA replication system in vitro. Results indicated that elongation reaction has been inhibited by UV irradiation while initiation reaction was not inhibited severely.
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