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Mechanism of rejoining of double-strand breaks in DNA in mammalian cells

哺乳动物细胞中DNA双链断裂的重新连接机制

基本信息

批准号：
10216206
负责人：
TACHIBANA Akira
金额：
$ 19.9万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2002
项目状态：
已结题

项目摘要

Repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. To investigate the process of NHEJ, we have established an in vitro system to clarify the measurement and analysis of the efficiency and the fidelity of rejoining of DSBs, and applied the method to investigate NHEJ in human cells derived from patients suffering from cancer-prone hereditary diseases. The efficiency of rejoining in the nuclear extract from an ataxia-telangiectasia (A-T) cell line was comparable to that from a control cell line. However, the accuracy of rejoining was much lower for the A-T cell extract than for the control cell extract. All mutations were deletions, most of which contained short direct repeats at the breakpoint junctions. The deletion spectrum caused by the A-T nuclear extract was distinct from that by the control extract. These results indicate that A-T cells have certain deficiencies in end-joining of double-strand breaks in DNA. The extract from Bloom syndro … More me cells also showed the similar activity and the lower fidelity of rejoing compared to that from normal cells. From the sequencing analysis of the junction o. DSBs, it is speculated that the defect in the BLM helicase might cause irregular rejoining of DSBs. Radioadaptive response is the acquirement of cellular resistance to ionizing radiation by prior exposure to low dose. We investigated the in vitro end-joining activity of DNA ends in radioadaptive cells. Both the efficiency and the fidelity of rejoining in the cells pre-exposed to low dose are increased comparing to those without pre-exposure. We also found that in p53-deficient cells, pre-irradiation caused no apparent alteration in both the efficiency and fidelity of end-joining. These results suggest that the exposure to low dose activates a cellular function to repair DSBs efficiently, which is dependent on p53. These results indicate that NHEJ pathway is regulated by many factors ; genetic regulation by ATM and BLM, and physiological conditions such as irradiation with ionizing radiation. The observations also suggest that in some occasions p53 might play a key role in NHEJ. Less

修复dsb对于防止染色体断裂、易位和缺失是很重要的。为了研究NHEJ的过程，我们建立了体外系统，明确了dsb再连接效率和保真度的测量和分析，并将该方法应用于易发遗传性疾病患者的人类细胞中NHEJ的研究。从共济失调毛细血管扩张（a-t）细胞系提取的核提取物中重新连接的效率与对照细胞系相当。然而，与对照细胞提取物相比，A-T细胞提取物的再连接准确性要低得多。所有突变都是缺失，其中大多数在断点连接处包含短的直接重复。A-T核提取物引起的缺失谱与对照提取物引起的缺失谱不同。这些结果表明，A-T细胞在DNA双链断裂的末端连接上存在一定的缺陷。与正常细胞相比，更多的me细胞也表现出相似的活性，但重新激活的保真度较低。从对dsb连接处的测序分析推测，BLM解旋酶的缺陷可能导致dsb的不规则再连接。辐射适应反应是指细胞通过先前的低剂量电离辐射照射而获得对电离辐射的抵抗力。我们研究了放射适应细胞DNA末端的体外末端连接活性。与未预暴露的细胞相比，低剂量预暴露细胞的再连接效率和保真度均有所提高。我们还发现，在p53缺陷细胞中，预辐照对末端连接的效率和保真度没有明显的改变。这些结果表明，低剂量暴露激活了细胞功能，有效地修复dsb，这依赖于p53。这些结果表明NHEJ通路受多种因素调控；ATM和BLM的基因调控，以及电离辐射照射等生理条件。观察结果还表明，在某些情况下，p53可能在NHEJ中起关键作用。少