1986 Fiscal Year Final Research Report Summary
Peconstitution and analysis of mammalian DNA replication system in vitro.
| Project/Area Number |
60571034
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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 | Tokyo University |
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Principal Investigator |
ENOMOTO Takemi Faculty of Pharmaceutical Sciences, University of Tokyo, Assistant professor., 薬学部, 助手 (80107383)
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| Project Period (FY) |
1985 – 1986
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| Keywords | DNA replication / in vitro reconstitution / DNA polymerase <alpha> / Primase / DNA helicase / 活性促進因子 |
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| Research Abstract |
In order to understand the molecular mechanism of DNA replication in mammalian cells, we have tried biochemical approaches to reconstitute and analyze several steps of DNA replication in vitro as well as genetic approaches to isolate and characterize DNA temperature-sensitive mutants. We have purified from mouse FM3A cells enzymes and proteins related to DNA replication such as DNA polymerase <alpha> -primase complex, DNA topoisomerases <I> and <II> , four DNA-dependent ATPases, and a factor stimulating DNA polymerase <alpha> activity.
1. The step to unwind double-stranded DNA.
We have tried to detect DNA helicase activity which is thought to participate in this step of DNA replication, and found the activity in one of the four DNA-de-pendent ATPases, ATPase B. It has been revealed that the helicase activity is dependent on the hydrolysis of ATP, and the enzyme can unwind up to about 120 base-long DNA.
2. The step to synthesize Okazaki fragments on single-stranded DNA.
We have established the conditions to dissociate and reconstitute the DNA poymerase <alpha> complex and analyzed by using the above technique the mechanism to determine the size of RNA primers and to switch primer RNA synthesis to DNA synthesis. It has been revealed that the complex formation between DNA polymerase <alpha> and primase and the existence of deoxyribonucleoside triphosphates play very important roles in the above processes.
3. The step to synthesize DNA on single-stranded DNA.
We have characterized a factor stimulating DNA polymerase <alpha> activity and indicated that the stimulation by the factor is due to the increase in the initiation frequency of DNA synthesis from the primers.
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