Title

探討 KAP1 在 DNA 損害反應中的角色

Translated Titles

The role of KAP1 in DNA damage response

Authors

林柏穎

Key Words

KAP1 ; DNA 損害反應 ; DNA 損害反應因子 ; micronuclei ; KAP1 ; DNA damage response ; DNA damage response factor ; micronuclei

PublicationName

中興大學分子生物學研究所學位論文

Volume or Term/Year and Month of Publication

2012年

Academic Degree Category

碩士

Advisor

楊文明

Content Language

繁體中文

Chinese Abstract

在細胞中,DNA 損害若發生在重要的基因上,會影響細胞正常功能,甚至癌化。為了避免這類情形發生,細胞藉由 DNA 損害反應機制 (DNA damage response, DDR),聚集 DNA 損害反應因子 (DDR factors) 在受損的 DNA 上,形成 repair foci,進而啟動 DNA 修復。近年來研究發現 KRAB-associated protein 1 (KAP1),以 S824 磷酸化的形式存在於 repair foci 中。但是,目前對於 KAP1 出現在 repair foci 的角色仍不清楚。根據研究顯示,當 DNA 損害發生時,若是剔除 KAP1,會降低 DNA 損害反應因子-BRCA1 聚集在 repair foci 的比例。由於 DNA 損害反應因子的聚集,主要由 mediators 吸引,而 KAP1 在基因轉錄中,也具有類似吸引的能力。因此,在本篇論文中,推測 KAP1 在 repair foci 中可能作為 mediator。並針對此點,探討 KAP1 是否吸引 DNA 損害反應因子,進而促進 DNA 修復的進行。 為了探討 KAP1 座落在 repair foci 是否普遍存在於各種不同 DNA 損害反應,我們藉由 Ultraviolet irradiation 和 micronuclei (MN) 作為 DNA 損害平台,觀察 KAP1 的分佈情形。結果顯示 KAP1 的確會座落在 repair foci 上。而且,含有 KAP1 的 MN 具有較低的 γH2AX 出現率。暗示 KAP1 可能在 MN 中,扮演某種角色來幫助 MN 進行 repair。為了進一步探討 KAP1 是否吸引 DNA 損害反應因子到 repair foci,藉由免疫共沉澱法的結果顯示,KAP1 與 53BP1、BRCA1、BARD1、MMS21 產生交互作用,暗示這四個 DNA 損害反應因子可能被 KAP1 吸引至 repair foci 中。進一步利用 KAP1dC bodies 和 MN 作為平台,觀察 KAP1 是否具有吸引 DNA 損害反應因子的能力。免疫螢光染色的結果顯示,KAP1 會吸引 53BP1、BARD1、BRCA1 和 MMS21 進入 repair foci 的能力。為了探討 KAP1 吸引 DNA 損害反應因子的能力是否有助於 DNA 修復,大量表現 KAP1 使 MN 含有 γH2AX 的比例有下降的趨勢。另外,利用流式細胞儀偵測人工製造的雙股斷裂系統所產生的 GFP 比例顯示,模擬 KAP1 knockdown 的突變-RV487488EE,會降低細胞表現 GFP 的比例,顯示 KAP1 有助於 DNA repair 的進行。 本篇論文研究發現:KAP1 座落在不同 DNA 損害所產生的 repair foci 上。進而吸引 53BP1、BARD1、BRCA1、MMS21 到 repair foci。而 KAP1 吸引 DNA 損害反應因子的能力有助於 DNA 修復的進行。

English Abstract

In cells, DNA damage in important genes can impair cell’s function and even promote tumorigenesis. In order to resolve this problem, DNA damage response (DDR) is turned on and DDR factors form repair foci at broken DNA. Subsequently, DNA repair is switched on to repair DNA. In recent years, Serine 824 (S824) phosphorylation at KRAB-associated protein 1 (KAP1) was found to accumulate at repair foci. Knockdown of KAP1 is also reported to decrease the accumulation frequency of one DDR factor, BRCA1, at repair foci. However, the role of KAP1 in DNA repair is still unclear. In the DDR pathway, mediators recruit DDR factors into repair foci. In analogy, KAP1 also contains similar recruiting ability in transcription regulation. In this study, we want to investigate if KAP1 can function as a mediator to recruit DDR factors into repair foci and help DNA repair. To investigate whether KAP1 locates at various DNA damage induced repair foci, we tested Ultraviolet irradiation and micronuclei (MN) as DNA damage platform. We observed that KAP1 co-localized with repair foci. In order to explore if KAP1 recruited DDR factors into repair foci, we performed co-immunoprecipitation (Co-IP) and found that KAP1 interacted with 53BP1, BARD1, BRCA1 and MMS21. The results suggest that these four DDR factors may be recruited to repair foci by KAP1. To prove this hypothesis, we used KAP1dC bodies and MN as a platform and tested if KAP1 had the ability to recruit DDR factors. The immunofluorescence results showed that KAP1 recruited 53BP1, BARD1, BRCA1 and MMS21 into repair foci. In order to verify if KAP1 recruiting ability was helpful for DNA repair, we found that over-expressed KAP1 decreased the percentage of MN containing γH2AX. Also, KAP1 RV487488EE, which mimicked knockdown mutant, decreased the GFP expression from an artificial DNA double strand break system. These results show that KAP1 recruiting ability helps DNA repair. Taken together, these results demonstrate that KAP1 can locate at different kinds of DNA damage induced repair foci. Moreover, KAP1 can recruit 53BP1, BRCA1, BARD1 and MMS21 into repair foci. And KAP1 recruiting ability is helpful for DNA repair.

Topic Category 生命科學院 > 分子生物學研究所
生物農學 > 生物科學
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