| Project/Area Number |
13672375
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Human genetics
|
|---|
| Research Institution | KYOTO UNIVERSITY |
|---|
Principal Investigator |
YAMAZOE Mitsuyoshi Kyoto Univ. Graduate School of Med. (Assistant Prof.), 医学研究科, 助手 (00284745)
|
|---|
| Project Period (FY) |
2001 – 2002
|
| Project Status |
Completed (Fiscal Year 2002)
|
| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
|
|---|
| Keywords | BRCA2 / homologous DNA recombination / chromosome stability / DT40 / conditional mutant / Cre-loxP recombination syste / 条件特異株 / Rad51パラログ / gene targeting / コンディショナル変異体細胞 / Cre-loxP組換えシステム |
|---|
| Research Abstract |
1. Mutant BRCA2 alleles used in this study
We have constructed the following mutant BRCA2 alleles: (a) complete null allele (null), (b) conditional null allele (con), c mutant alleles which lack different sizes of C terminal region in BRCA2
2. Phenotype of a complete null mutant of BRCA2
Elimination of the BRCA2 gene in a conditional null mutant (null/con) caused cell growth arrest and cell death, indicating that BRCA2 is essential for normal cell growth
3. Analysis of hypomorphyic mutants of BRCA2
We have established the following mutants partially deficient in BRCA2 function
(a) A mutant cell in which a targeted mutation is predicted to truncate treBRCA2 protein between BRC repeant 3 and 4 (BRC3)
(b) Amutant cell expressing BRCA2 in which nuclear localization signals are deleted (ΔNLS)
<Phenotypes of the BRC3 and ΔNLS mutants>
(1) Growth properties
Although the ΔNLS mutant cell can grow as well as a wild-type cell, the growth rate of the BRC3 mutant was significantly lower than of wildtype cel
… More
ls
(2) Sensitivity to DNA damage
The ΔNLS mutant cells showed higher sensitivity to the DNA cross-linking agent cisplatin and the BRC3 mutant cells showed higher sensitivity to both ionizing radiation and cisplatin, compared with wild-type cells
(3)Rad51 focus formation after genotoxic treatment
We found that the formation of Rad51 foci was severely impaired in the BRC3 mutant following γ irradiation
(4) Efficiency of homologous recombination
The BRC3 mutant exhibited significantly reduced efficiency of both Ig gene conversion and targeted integration
These observations suggest that BRCA2 is involved in homologous DNA recombination and enhances recruitment of Rad51 to damaged DNA sites
4. Future prospects
(1) In order to elucidate function of conserved regions existed in both N- and C- terminal regions of BRCA2, we plan to generate BRCA2 mutants in which the corresponding regions are missing
(2) In order to clarify epistasis between BRCA2 and Rad51 paralogs, we plan to generate double mutants containing BRC3 mutation in BRCA2 and null mutation in Rad51 paralogs Less
|
