2007 Fiscal Year Final Research Report Summary
Analysis of DNA strand break repair using human knockout cells
| Project/Area Number |
18590063
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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 |
Biological pharmacy
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| Research Institution | Yokohama City University |
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Principal Investigator |
ADACHI Noritaka Yokohama City University, International Graduate School of Arts and Sciences, Associate Professor (30264675)
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| Project Period (FY) |
2006 – 2007
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| Keywords | DNA strand break / DNA repair / end-ioining / random integration / gene targeting / nonhomologous recombination |
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| Research Abstract |
We recently developed a system using the human cell line Nalm-6 that enables rapid production of human gene knockout cells. In this study, we used this system to genetically analyze the function of human genes that have been implicated in DNA strand break repair. Specifically, we disrupted a series of genes involving those for Rad54, FancB, Mus81, Tdp1, Ku70/80, and Lig3, which all have some roles in DNA single and/or double-strand break repair in human cells. In addition, we tried to produce as many double-knockout mutant cells as possible, particularly those with a Lig4 mutation. Using these mutants, we have successfully analyzed the cellular sensitivity to anticancer agents involving cisplatin and topoisomerase inhibitors as well as the genetic interaction between p53 and BLM and between Mus81 and FancB, We also performed gene knockdown experiments with siRNAs for DNA ligases that are potentially involved in random integration and/or gene targeting. The results suggested that suppressing nonhomologous end-joining reactions could enhance the efficiency of gene targeting. Our findings also indicated that there must be another mechanism for random integration that does not rely on the nonhomologous end-joining pathway.
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