Reverse Genetic Analysis of Homologous DNA Recombination in Higher Eukaryotes

高等真核生物中同源 DNA 重组的反向遗传分析

• 批准号: 10480192
• 负责人: TAKEDA Shunichi
• 金额: $ 7.74万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10480192/
• 关键词: Homologous DNA recombination; Rad 51; DT40; DNA repair; ジーンターゲティング; ヌクレオチド除去修復; 相同DNA組換え; 標的組換え; ターゲットインテグレーション; 放射線; 染色体断裂

Chromosomal double-stand breaks (DSBs) arise after ionizing irradiation and during DNA replication. A chromosomal break is a lethal event, if it left unrepaired. There are two major DSB repair pathways, homologous DNA recombination (HR) and nonhomologous DNA end-joining, which are highly conserved from yeast to mammals. Rad51, which is structurally and functionally related to the Escherichia coli recombination protein RecA, Rad51B, Rad54 and Mre11 are involved in HR, while the Ku proteins are involved in end-joining. HR-deficient. yeast cells are capable of proliferating, though murine cells deficient in Rad51 or Mre11 are not viable. To investigate the essential roles of HR in vertebrate cells, we have generated conditionally Rad51-and Mre11-deficient cells from hyper-recombinogenic chicken B lymphocyte line DT40. The depletion of Rad51 or Mre11 caused the appearance of chromosomal breaks and subsequent cell death. These observations indicate that HR is required to repair spontaneousl … More y-arising DSBs, possibly during DNA replication. To further analyze the involvement of HR in repairing spontaneous DNA lesions, we measured the level of sister chromatid exchange (SCE) in HR-deficient cells, which is induced by crosslinking agents and known to be intimately associated with DNA replication. The levels of both spontaneous and induced SCE were strongly reduced in HR-deficient cells including RAD51ィイD1-/-ィエD1, Rad51BィイD1-/-ィエD1 and RAD54ィイD1-/-ィエD1 cells, but not in KU70ィイD1-/-ィエD1 cells, confirming that HR is indeed the mechanism responsible for SCE. HR may use the nascent sister chromatid to repair potentially lethal DNA lesions accomp anying replication. Since crossing-over is a relatively infrequent event in yeast mitosis, the presence of as many as 1-5 SCEs (i. e., visible cross-overs) implies that recombinational rep air without such crossovers may also occur frequently in the cycling human cells. Conceivably, HR has to play a more important role in maintaining chromosomal DNA in vertebrate cells than it does in yeast, probably due to the several hundred-fold difference in genome size between vertebrates and lower eukaryotes. Less

染色体双链断裂（DSB）发生在电离辐射后和DNA复制过程中。染色体断裂如果不加以修复，将是致命的。存在两种主要的DSB修复途径，同源DNA重组（HR）和非同源DNA末端连接，其从酵母到哺乳动物高度保守。Rad 51在结构和功能上与大肠杆菌重组蛋白RecA相关，Rad 51 B、Rad 54和Mre 11参与HR，而Ku蛋白参与末端连接。人力资源不足酵母细胞能够增殖，尽管缺乏Rad 51或Mre 11的鼠细胞不能存活。为了研究HR在脊椎动物细胞中的重要作用，我们从超重组鸡B淋巴细胞系DT 40中产生了条件性Rad 51和Mre 11缺陷细胞。Rad 51或Mre 11的缺失导致染色体断裂和随后的细胞死亡。这些观察结果表明，HR需要自发修复 ...更多信息 y产生的DSB，可能在DNA复制期间。为了进一步分析HR参与修复自发性DNA损伤，我们测量了HR缺陷细胞中的姐妹染色单体交换（SCE）水平，这是由交联剂诱导的，已知与DNA复制密切相关。自发和诱导SCE的水平在HR缺陷细胞中均显著降低，包括RAD 51 β-D1-/-β-D1、RAD 51 β-D1-/-β-D1和RAD 54 β-D1-/-β-D1细胞，但在KU 70 β-D1-/-β-D1细胞中没有，证实HR确实是SCE的机制。HR可能利用新生的姐妹染色单体来修复伴随复制的潜在致死性DNA损伤。由于交换是酵母有丝分裂中相对罕见的事件，因此存在多达1-5个SCE（即，例如，可见的交换）意味着没有这种交换的重组REPAIR也可能频繁地发生在循环的人细胞中。可以想象，HR在维持脊椎动物细胞中的染色体DNA方面比在酵母中发挥更重要的作用，这可能是由于脊椎动物和低等真核生物之间基因组大小的数百倍差异。少

项目成果

Morrison C.,Shinohara A.,Sonoda E.,Yamaguchi-Iwai Y.,Takata M.,Weichselbaum R.R.,Takeda S.: "The Essential functions of human Rad 51 are independent of ATP hydrolysis"Mol Cell Biol. 19. 6891-6897 (1999)

Morrison C.、Shinohara A.、Sonoda E.、Yamaguchi-Iwai Y.、Takata M.、Weichselbaum R.R.、Takeda S.：“人类 Rad 51 的基本功能独立于 ATP 水解”Mol Cell Biol。

Sonoda E, Sasaki M S, Morrison C, Yamaguchi・Imai Y, Takata M, Takada S: "Sister Chromatid exchanges are mediated by homologous recombination in varte brate cells"Mol Cell Bio. 19. 5166-5169 (1999)

Sonoda E、Sasaki MS、Morrison C、Yamaguchi·Imai Y、Takata M、Takada S：“姐妹染色单体交换是由 varte brate 细胞中的同源重组介导的”Mol Cell Bio. 19. 5166-5169 (1999)

N.Takao, H.Kato, R.Mori, C.Morrison, E.Sonoda, X.Sun, H.Shimizu, K.Yoshioka, S.Takeda and K.Yamamoto: "Disruption of Atm in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation."Oncogene.. 18. 7002-7009 (1999)

N.Takao、H.Kato、R.Mori、C.Morrison、E.Sonoda、X.Sun、H.Shimizu、K.Yoshioka、S.Takeda 和 K.Yamamoto：“p53 缺失细胞中 Atm 的破坏导致

M.Takata, M.S.Sasaki, E.Sonoda, T.Fukushima, J.Albala, S.Swagemakers, R.Kanaar, L.H.Thompson and S.Takeda: "Targeted disruption of the RAD51B, amember of RAD51-related gene family, impairs homologous recombination and repair of crosslink DNA damages."Mol.

M.Takata、M.S.Sasaki、E.Sonoda、T.Fukushima、J.Albala、S.Swagemakers、R.Kanaar、L.H.Thompson 和 S.Takeda：“RAD51B（RAD51 相关基因家族的成员）的靶向破坏会损害

E.Sonoda et al.: "Rad51 deficient vertebrate cells accumulate chromosomal breaks prior to cell death." EMBO J.vol.17(2). 598-608 (1998)

E.Sonoda 等人：“Rad51 缺陷的脊椎动物细胞在细胞死亡之前会积累染色体断裂。”