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; 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

电离照射和DNA复制期间出现染色体双固定断裂（DSB）。如果未修复，染色体断裂是致命的事件。有两种主要的DSB修复途径，同源DNA重组（HR）和非整体DNA端结合，它们从酵母到哺乳动物都高度保守。 RAD51在结构和功能上与大肠杆菌重组蛋白RECA，RAD51B，RAD54和MRE11相关，而KU蛋白参与了最终结合。 HR缺乏。酵母细胞能够增殖，尽管鼠类细胞在RAD51或MRE11中不足。为了研究人力资源在脊椎动物细胞中的基本作用，我们从高品质组成鸡B淋巴细胞dt40的有条件地产生了RAD51和MRE11缺陷型细胞。 RAD51或MRE11的耗竭导致染色体断裂的出现和随后的细胞死亡。这些观察结果表明，在DNA复制过程中可能需要HR修复赞助的dsbs。为了进一步分析HR在修复赞助DNA病变中的参与，我们测量了HR缺陷型细胞中姊妹染色单体交换（SCE）的水平，该细胞是通过交联剂引起的，并已知与DNA复制密切相关。在HR缺陷的细胞中，赞助商和诱导的SCE的水平均大大降低，包括RAD51D1 - / - IE D1，RAD51BIY D1 - / - IE D1和RAD54D1 - / - IE D1细胞，但在KU70D1 - / - IE D1细胞中却不是对HR的责任。人力资源部可能会使用新生的姐妹染色单体修复潜在的致命DNA病变会经历任何复制。由于越过酵母有丝分裂中的跨越是一个相对频繁的事件，因此存在多达1-5的SCE（即可见的跨界），这意味着在没有这种交叉交叉的情况下，重组rep空气也可能经常发生在循环的人类细胞中。可以想象，人力资源在维持脊椎动物细胞中的染色体DNA中必须发挥更重要的作用，这可能是由于脊椎动物和较低的真核生物之间的基因组大小几百倍差异所致。