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dc.description.abstractThe breast cancer susceptibility gene BRCA2 has been implicated in transcription, DNA repair and cell cycle (McKeon, 1999; Venkitaraman, 2001; Wooster et al., 1995). However, our understanding about the functions of BRCA2 is far from satisfaction. BRCA2 interacts directly with RAD51 (Sharan et al., 1997), which is a mammalian homologue of the bacterial protein RecA involved in homologous recombination (HR) (Khanna and Jackson, 2001; Sonoda et al., 1998; van Gent et al., 2001). Eight BRC repeats in BRCA2 prevent RAD51 from polymerizing by mimicking RAD51 polymerization motif, and recruit RAD51 to DNA damaged site (Davies et al., 2001; Pellegrini et al., 2002; Shin et al., 2003). Mice disrupted with Brca2 allele undergo embryonic lethality (Ludwig et al., 1997; Suzuki et al., 1997), indicating that Brca2 may function in embryonic development. However, mice of which Brca2 allele was disrupted in exon 11 (Brca2^(Tr/Tr)) exhibit prolonged embryonic survival, increased DNA damage sensitivity, growth retardation and chromosome aberrations (Connor et al., 1997; Patel et al., 1998). These suggest that Brca2 may play a crucial role in DNA repair. Paradoxically, Brca2^(Tr/Tr) mice (0.8% birth rate) die from thymic lymphomas in 12 weeks after birth (Connor et al., 1997; Friedman et al., 1998). Murine embryonic fibroblast cells (MEFs) isolated from Brca2^(Tr/Tr) mice undergo progressive cell cycle arrest in the G1 and G2 phases, in agreement with the embryonic lethality and growth retardation(Lee et al., 1999; Patel et al., 1998). Indeed, progressive growth failure is due to the activation of cell cycle checkpoint, by the accumulation of aberrant DNA adducts. It has been suggested previously, that Brca2 mutation cooperates with the mutation in Bub1, BubR1, mitotic checkpoint proteins and p53 in the generation of chromosomal instability (Lee et al., 1999). But the direct contribution of mitotic checkpoint in tumorigenesis has been unclear so far. Here, I have attempted to demonstrate that functional and physical interaction between BRCA2 and mitotic checkpoint is crucial in the maintenance of genetic integrity. I find that BRCA2 interacts physically with BubR1, a component of mitotic checkpoint in transfected 293T cells. To explore the physiological meaning of the interaction between BRCA2 and BubR1, the possibility of BubR1 in DNA damage response was tested by adopting RNA interference technology. This idea is based on the assumption that if mitotic checkpoint is involved in tumorigenesis, it is very likely to be involved in DNA damage response, especially in Brca2 mutated cells. Transient silencing of BubR1 expression induces cells to accumulate in the S and G_(2) phases upon DNA damage. Strikingly, the protein level of BubR1 is stabilized in response to double stranded DNA break. In contrast, western analysis shows that the level of BubR1 is unchanged in Brca2-deficient cells. Furthermore, BubR1 level is decreased in Chk2-inactivated cell line. Taken together, these results imply that BubR1, previously defined as a mitotic checkpoint protein, is also involved in DSB (Double Stranded DNA Break) response pathway downstream of Chk2 and BRCA2. ;유방암 억제 유전자 BRCA2는 전사, DNA 복구, 세포주기와 관련되어있다. 그러나 BRCA2의 기능이 완전히 밝혀진 것은 아니다. BRCA2는 homologous recombination에 중요한 기능을 담당하는 RecA의 homologue인 RAD51과 직접 결합한다. 특히, BRCA2의 8개의 BRC repeats는 RAD51의 motif를 모방하여 그들의 polymerization을 방해, DNA 복구가 원활히 일어나도록 도와준다. Brca2의 Knock-out mice는 배 발생 중에 사망하게 되는데, 이는 Brca2가 배 발생에 중요한 역할을 할 것을 시사한다. 그러나 Exon11을 포함한 3' region을 truncation 시킬 경우, DNA손상에 민감하고, 염색체 이상과 성장 지연을 보이는 mice가 낮은 생존율 (0.8%)로 태어나게 된다. 이는 Brca2가 DNA 복구에 중요할 역할을 할 것을 뒷받침하고 있다. 그런데 모순적이게도, 성장 지연을 보이는 Brca2^(Tr/Tr) mice는 태어난 후, 12주 만에 thymic lymphoma를 일으켜 사망하게 된다. Murine embryonic fibroblast 세포 (MEFs)는 proliferation됨에 따라 G1, G2 기에 세포주기가 멈추게 된다. 참으로 성장 지연은 손상된 DNA가 축적되어 세포주기 checkpoint가 작동하면서 나타난 결과이다. Brca2의 돌연변이는 Bub1, BubR1, p53 등의 돌연변이와 협조적으로 chromosomal instability를 나타낸다. 그러나 mitotic checkpoint가 암 발생에 기여하는지는 아직 명확하지 않다. 이에 논문을 통해, BRCA2와 mitotic checkpoint 사이의 상호협력이 genetic integrity를 유지하는데 중요할 것인지를 밝히고자 하였다. 먼저, 293T 세포에서 BRCA2와 mitotic checkpoint 단백질인 BubR1과의 결합을 확인하였다. 다음으로 RNA interference technology를 이용하여, BRCA2와 BubR1의 상호결합이 DNA damage response에 관여하는지 조사하였다. 이는 만약 mitotic checkpoint가 암 발생에 기여한다면, DNA damage response와 연결되어야 한다는 가정하에 시도된 것이다. BubR1의 transient silencing은 S와 G2기에 세포들이 축적됨을 보였다. 또한 놀랍게도, BubR1의 단백질 수준이 DNA double stranded break가 발생했을 때, 안정되는 것을 볼 수 있었다. 반면, Brca2^(Tr/Tr) MEFs에서는 BubR1의 단백질의 안정화를 관찰할 수 없었다. 나아가, DNA damage response pathway의 중요한 구성원인 Chk2가 inactivation되었을 때에 BubR1은 오히려 감소되었다. 이러한 결과들은 BubR1이 BRCA2와 Chk2를 통해 DNA damage response에 관여하고 있음을 보여준다.-
dc.description.tableofcontentsAbstract = iv ㆍ Introduction = 1 1. Functions of Tumor suppressor BRCA2 = 1 2. Mitotic check point in the maintenance of Genomic Integrity = 5 3. The functions of BubR1 = 8 ㆍ Materials and Methods= 2 1. Cell Culture = 12 2. Transfection = 12 3. Transduction = 12 4. Immunoprecipitation and Immunoblotting = 13 5. Gene silencing by Small Interfering RNA = 14 6. Cell Cycle analysis = 15 7. Treatment = 15 8. Instruments = 16 ㆍ Results = 17 1. BRCA2 interacts with BubR1 physically in 293T cells = 17 2. Physical interaction of BRCA2 and BubR1 may function in cellular response to DNA damage = 19 3. Bub1 affects cell cycle progression upon DNA damage = 24 4. BubR1 responds to DNA damage signal = 26 5. Putative involvement of BubR1 in DNA damage pathway through either BRCA2 or Chk2 = 30 ㆍ Discussion = .34 ㆍ References = 39 ㆍ 논문개요 = 49 감사의 글 = 51-
dc.format.extent551148 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleBubR1, a mitotic checkpoint protein, responds to DNA damage through BRCA2 and Chk2-
dc.typeDoctoral Thesis-
dc.format.pagev, 53 p.-
dc.identifier.major대학원 분자생명과학부- 2-
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