蛋白KIAA1751和XLF的功能研究
摘要
真核生物对维持基因组的稳定性及防止非整倍体的产生,有其一套完整的保护机制,即细胞周期调控、检验点调控以及对损伤DNA的修复。当细胞感应到DNA损伤、复制应力或有丝分裂异常,招募和激活其下游的信号转导分子和效应分子,延迟细胞进入细胞周期的下一个阶段。DNA双链损伤(DSB)是已知的最严重的细胞损伤,只要有一处未被修复的DSB就能引发程序性细胞凋亡。同源重组(HR)和非同源末端连接(NHEJ)是细胞内最主要的DSB修复途径。本论文分为两个部分:
(1)KIAA1751是一个含有ASH结构域的蛋白。含有这个结构域的蛋白通常与纤毛、鞭毛、中心体、以及高尔基体有关,并具有结合微管的功能。本文发现KIAA1751蛋白在细胞内主要定位在细胞核,ASH结构域突变体(N475A)对细胞内定位没有明显变化。抑制KIAA1751的表达导致紫杉醇诱导的纺锤体装配检验点激活的缺陷,提示KIAA1751参与纺锤体装配检验点的调控。
(2)NHEJ核心因子XLF通过与XRCC4-连接酶Ⅳ复合物中的XRCC4结合,来稳定并激活DNA连接酶Ⅳ对损伤DNA末端的连接活性;Werner综合症中缺陷的蛋白WRN既有解旋酶活性,又有外切核酸酶活性。在XRCC4-连接酶Ⅳ复合物与损伤DNA末端连接之前,WRN可以通过结合XRCC4来与这一复合物相互作用,激活其自身的外切核酸酶活性,使得其在NHEJ过程中作为DNA末端加工因子行使核酸酶功能。我们验证了抑制XLF的表达导致DNA损伤修复的缺陷,发现XLF自身和WRN对XLF的转录有正调节作用。
To ensure genome integrity and prevent aneuploidy,eukaryotes have evolved an integrated protective mechanism,including cell cycle control,checkpoint control,and DNA repair.When a cell senses DNA damage,replication stress,or abnormal mitosis, it recruits and activates a series of signal transducers and effectors,thus halting from entering into the next ophase of the cell cycle.DNA double-strand break(DSB) is probably the most lethal attack,with as little as one unrepaired DSB being capable of triggering programmed cell death.Homologous recombination(HR) and nonhomologous end-joining(NHEJ) are the major cellular DSB repair pathways.This thesis research contains two parts:
(1) KIAA1751 is an ASH domain-containing protein.This domain is present in proteins associated with cilia,flagella,the centrosome,and the Golgi complex,and possesses a microtubule-binding function.This research has showed that KIAA1751 mainly locates in the nucleus,mutation of its ASH domain does not affect its localization.We have also found that depletion of KIAA1751 by siRNA leads to a defective spindle checkpoint activation induced by taxol,indicating that KIAA1751 is involved in the spindle checkpoint control.
(2) By interacting with the XRCC4-DNA ligase IV complex,the NHEJ core factor XLF stabilizes and stimulates the DNA ligase to the damaged DNA.The human RecQ family helicase WRN,which is mutated in the Werner syndrome, possesses both helicase and 3'-5' exonuclease activities.Before the ligation of XRCC4-DNA ligase IV complex and damaged DNA terminal,WRN interacts with this complex by binding to XRCC4.Here we have confirmed that depletion of XLF leads cellular defects in DNA repair,and have found that XLF itself along with WRN positively regulates the transcription of XLF.
(1)KIAA1751是一个含有ASH结构域的蛋白。含有这个结构域的蛋白通常与纤毛、鞭毛、中心体、以及高尔基体有关,并具有结合微管的功能。本文发现KIAA1751蛋白在细胞内主要定位在细胞核,ASH结构域突变体(N475A)对细胞内定位没有明显变化。抑制KIAA1751的表达导致紫杉醇诱导的纺锤体装配检验点激活的缺陷,提示KIAA1751参与纺锤体装配检验点的调控。
(2)NHEJ核心因子XLF通过与XRCC4-连接酶Ⅳ复合物中的XRCC4结合,来稳定并激活DNA连接酶Ⅳ对损伤DNA末端的连接活性;Werner综合症中缺陷的蛋白WRN既有解旋酶活性,又有外切核酸酶活性。在XRCC4-连接酶Ⅳ复合物与损伤DNA末端连接之前,WRN可以通过结合XRCC4来与这一复合物相互作用,激活其自身的外切核酸酶活性,使得其在NHEJ过程中作为DNA末端加工因子行使核酸酶功能。我们验证了抑制XLF的表达导致DNA损伤修复的缺陷,发现XLF自身和WRN对XLF的转录有正调节作用。
To ensure genome integrity and prevent aneuploidy,eukaryotes have evolved an integrated protective mechanism,including cell cycle control,checkpoint control,and DNA repair.When a cell senses DNA damage,replication stress,or abnormal mitosis, it recruits and activates a series of signal transducers and effectors,thus halting from entering into the next ophase of the cell cycle.DNA double-strand break(DSB) is probably the most lethal attack,with as little as one unrepaired DSB being capable of triggering programmed cell death.Homologous recombination(HR) and nonhomologous end-joining(NHEJ) are the major cellular DSB repair pathways.This thesis research contains two parts:
(1) KIAA1751 is an ASH domain-containing protein.This domain is present in proteins associated with cilia,flagella,the centrosome,and the Golgi complex,and possesses a microtubule-binding function.This research has showed that KIAA1751 mainly locates in the nucleus,mutation of its ASH domain does not affect its localization.We have also found that depletion of KIAA1751 by siRNA leads to a defective spindle checkpoint activation induced by taxol,indicating that KIAA1751 is involved in the spindle checkpoint control.
(2) By interacting with the XRCC4-DNA ligase IV complex,the NHEJ core factor XLF stabilizes and stimulates the DNA ligase to the damaged DNA.The human RecQ family helicase WRN,which is mutated in the Werner syndrome, possesses both helicase and 3'-5' exonuclease activities.Before the ligation of XRCC4-DNA ligase IV complex and damaged DNA terminal,WRN interacts with this complex by binding to XRCC4.Here we have confirmed that depletion of XLF leads cellular defects in DNA repair,and have found that XLF itself along with WRN positively regulates the transcription of XLF.
引文
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