DNA同源重组修复的分子机制

电离辐射直接造成生物靶分子细胞DNA的损伤，DNA的损伤类型很多，其中以DNA双链断裂（double strand break，DSB）最为严重。DNA DSB的修复较其他类型的DNA损伤更加困难。不修复则可能导致染色体断裂和细胞死亡，而修复不当则可能导致染色体缺失、重排、转位和倒置等，从而易于形成肿瘤等疾病。DNA损伤的不完全修复可导致基因组不稳定，机体细胞为了对抗损伤，发展出多个修复系统来保证基因组的完整性，同源重组修复（homologous recombination repair，HRR）是DNA DSB损伤修复的主要方式，对于保持哺乳动物细胞的基因组完整性十分重要。重组即遗传物质的重排，同源重组是指发生在同源DNA序列间的重组，主要是利用DNA序列间的同源性来识别，而负责配对和重组的蛋白质因子并无碱基序列特异性。     

DNA双链断裂修复途径中重要的修复蛋白

DNA双链断裂修复是DNA损伤最主要的修复途径之一,修复基因可以修复DNA损伤,保持遗传信息的完整性,从而抑制肿瘤的发生。目前已知参与DNA双链断裂损伤修复的机制有两种——非同源性末端连接和同源重组修复机制。该文介绍了参与非同源末端连接和同源重组修复机制的几种重要的修复蛋白。     

应用脉冲电场凝胶电泳分析X射线诱发人骨肉瘤细胞DNA双链断裂与损伤修复效应

目的 研究电离辐射诱发人骨肉瘤肿瘤细胞DNA双链断裂与辐射损伤修复效应, 观察辐射损伤、损伤修复效应与肿瘤细胞辐射敏感性之间的关系。方法 选用强制均匀电场电泳, 分别测定经不同剂量X射线照射和相同剂量照射后培养不同时间, 人骨肉瘤Rho0和143.B肿瘤细胞株DNA双链断裂。结果 (1)X射线诱发人骨肉瘤肿瘤细胞的DNA双链断裂与辐射剂量呈线性正比关系; (2)培养后的人骨肉瘤肿瘤细胞对辐射诱发的DNA双链断裂具有一定修复能力; (3)Rho0比143.B细胞株具有更高的辐射敏感性; (4)脉冲电场凝胶电泳技术是分析人肿瘤细胞DNA双链断裂的敏感方法。结论 脉冲电场凝胶电泳是分析人肿瘤细胞DNA双链断裂的敏感方法; 电离辐射诱发人骨肉瘤细胞DNA双链断裂与损伤修复效应和肿瘤细胞的辐射敏感性有密切关系。     

组蛋白去乙酰酶阻滞剂的放射增敏作用

放射治疗是肿瘤的重要治疗手段之一,辐射可以导致细胞DNA双链断裂。细胞主要通过同源重组修复和非同源末端连接修复方式修复DNA双链断裂。随着对双链DNA损伤修复机制认识的深化,组蛋白去乙酰酶（HDAC）阻滞剂成为提高放射敏感性的一种新策略。HDAC可分为4类。HDAC阻滞剂可非特异性地或特异性地阻滞这4类HDAC,使组蛋白乙酰化水平提高,染色体解螺旋,核小体结构改变。一方面使DNA更易受到辐射的影响;另一方面通过降低E2F1转录因子活性抑制损伤修复蛋白Ku80、Rad51等的表达,使其不能募集DNA损伤修复蛋白,且不能形成相应的蛋白复合物,使同源重组修复和非同源末端连接修复作用延缓,在伴有或不伴有肿瘤细胞凋亡增加的情况下,提高放射敏感性。现已有一些临床试验在进行中,并取得了初步的结果。     

γ-H2AX分析及其在监测电离辐射所致DNA双链断裂中的应用前景

近几年的研究表明,组蛋白H2AX在DNA损伤修复、细胞周期检查点调控、基因组稳定的维持和肿瘤抑制中起着重要作用,而且在放射生物学中具有应用前景。磷酸化的组蛋白H2AX (y-H2AX)对DNA双链断裂快速敏感的反应使其成为DNA双链损伤的标志。y-H2AX分析也将在监测电离辐射尤其是低剂量电离辐射所致的DNA双链损伤中拥有广阔的应用前景。     

DNA双链断裂残留损伤与癌细胞辐射敏感性研究

目的 了解不同组织来源癌细胞株和人体肿瘤组织原代细胞的DNA双链断裂损伤修复的个体差异性,探寻预测癌细胞辐射敏感性的生物指标。方法 ⁶⁰Co γ射线照射诱发DNA损伤,脉冲电场凝胶电泳检测DNA双链断裂损伤修复,细胞克隆形成能力法检测细胞辐射敏感性。结果 8个不同组织来源癌细胞株的辐射敏感性有较大的差异(D₀为0.65~2.15 Gy),不同细胞株20 Gy γ射线照射诱发产生的DNA双链断裂原初损伤有一定的差别,但与细胞辐射抗性无相关性。辐射敏感细胞SX-10的DNA双链断裂修复缺陷发生在早期快速修复相,而A2780细胞的修复缺陷是发生在晚期慢速修复相。20 Gy照射修复2 h后DNA双链断裂残留量与细胞辐射敏感性指标D₀或SF₂值有显著的相关性。不同个体患者脑肿瘤组织原代细胞之间,辐射诱发DNA双链断裂的修复反应存在明显差异,修复2 h后残留损伤的个体差异性分布类似于癌细胞株。结论 DNA双链断裂残留损伤与癌细胞辐射抗性有显著相关性,可作生物指标预测肿瘤组织细胞对放射治疗的反应性。     

AT细胞对电离辐射敏感原因的探讨

AT细胞具有对电离辐射和拟辐射物质的高敏感性以及DNA合成抑制市性，存在复杂的基因异质性，本从AT细胞的遗传学互补性分析，DNA损伤修复缺陷以及DNA拓扑酶学研究三方面对AT细胞辐射敏感性的分子机理等进行了讨论，认为DNA双链修复缺陷与重接忠实性下降可能是AT细胞电离辐射敏感性的原因。     

CUL4A-DDB1细胞稳定株筛选及DNA双链断裂模型构建

目的为了鉴定并验证CUL4A-DDB1泛素连接酶复合体中参与DNA损伤修复反应过程的1～2种关键成分在损伤识别、早期及晚期修复中的动态变化,拟构建含有串联亲和纯化（TAP）标签载体并筛选高表达CUL4A/DDB1的细胞株,建立DNA双链断裂模型。方法利用PCR获得CUL4A/DDB1基因,构建重组表达载体pNTAP-A-CUL4A/DDB1;使用顺铂和电离辐射刺激等外界刺激建立合适的DNA双链断裂细胞模型,使用G418筛选稳定表达CUL4A/DDB1的细胞株。结果与结论成功构建pNTAP-A-CUL4A/DDB1表达载体,建立合适的DNA双链断裂细胞模型,筛选得到稳定表达CUL4A/DDB1的细胞稳定株,为下一步质谱分析CUL4A-DDB1泛素连接酶在DNA损伤修复过程中的新功能打下基础。     

Establishment of CUL4A /DDB1 stable expression cell strain and cell models responding to DNA damage

目的 为了鉴定并验证CUL4A-DDB1泛素连接酶复合体中参与DNA损伤修复反应过程的1~2种关键成分在损伤识别、早期及晚期修复中的动态变化,拟构建含有串联亲和纯化(TAP)标签载体并筛选高表达CUL4A/ DDB1的细胞株,建立DNA双链断裂模型.方法 利用PCR获得CUL4A/DDB1基因,构建重组表达载体pNTAP-A-CUL4A/ DDB1;使用顺铂和电离辐射刺激等外界刺激建立合适的DNA双链断裂细胞模型,使用G418筛选稳定表达CUL4A/ DDB1的细胞株.结果 与结论 成功构建pNTAP-A-CUL4A/ DDB1表达载体,建立合适的DNA双链断裂细胞模型,筛选得到稳定表达CUL4A/ DDB1的细胞稳定株,为下一步质谱分析CUL4A-DDB1泛素连接酶在DNA损伤修复过程中的新功能打下基础.     

一种研究高等细胞内DNA双、单链断裂修复的新方法——病毒探针法

电离辐射引起的DNA双链断裂是细胞最重要的生物损伤之一。但是中性蔗糖沉降法及最近兴起的中性滤过洗脱法一般不适于哺乳动物双链断裂及细胞内修复的研究。作者发明了一种以病毒作为细胞内探针的方法来研究单、双链断裂。此类病毒是一种亲核的小型环状DNA病毒,自身缺乏DNA修复能力(papovaviruses SV40 and BK)。将受照病毒感染未受照细胞后分别进行不同时间的培养。分离核,提取病毒DNA并测定其剩余的损伤。此法用琼脂糖凝胶电泳分离病毒DNA,可见三条带,分别含双链断裂,单链断裂及无断裂的DNA分子。Southern blot法     

Formation and repair of clustered damaged DNA sites in high LET irradiated cells

Purpose: Radiation with high linear energy transfer (LET) produces clustering of DNA double-strand breaks (DSB) as well as non-DSB lesions. Heat-labile sites (HLS) are non-DSB lesions in irradiated cells that may convert into DSB at elevated temperature during preparation of naked DNA for electrophoretic assays and here we studied the initial formation and repair of these clustered damaged sites after irradiation with high LET ions.

Materials and methods: Induction and repair of DSB were studied in normal human skin fibroblast (GM5758) after irradiation with accelerated carbon and nitrogen ions at an LET of 125 eV/nm. DNA fragmentation was analyzed by pulsed-field gel electrophoresis (PFGE) and by varying the lysis condition we could differentiate between prompt DSB and heat-released DSB.

Results: Before repair (t = 0 h), the 125 eV/nm ions produced a significant fraction of heat-released DSB, which appeared clustered on DNA fragments with sizes of 1 Mbp or less. These heat-released DSB increased the total number of DSB by 30–40%. This increase is similar to what has been found in low-LET irradiated cells, suggesting that the relative biological effectiveness (RBE) for DSB induction will not be largely affected by the lysis temperature. After 1–2 hours repair, a large fraction of DSB was still unrejoined but there was essentially no heat-released DSB present.

Conclusions: These results suggest that high LET radiation, as low LET gamma radiation, induces a significant fraction of heat-labile sites which can be converted into DSB, and these heat-released DSB may affect both induction yields and estimates of repair.     

ADPRT抑制剂对人肿瘤细胞的放射增敏效应

目的：从细胞的克隆形成能力和细胞ＤＮＡ双链断裂及修复几方面探讨了ＡＤＰ－核糖基转移酶（ＡＤＰＲＴ）的特异性抑制剂３－氨基苯甲酰胺（３－ＡＢ）对人卵巢癌细胞株ＨＯＣ８的放射增敏效应。结果表明，３－ＡＢ能降低受照细胞的克隆形成能力；照射所诱发的初始ＤＮＡ双链断裂水平不受３－ＡＢ的影响，但细胞对双链断裂的修复能力受到抑制，表现为慢速修复水平下降，两方面的结果呈正相关。结论：通过脉冲电场凝胶电泳测定ＤＮＡ双链断裂及其修复水平，可以预测细胞的放射敏感性。     

Repair of the double-strand breaks induced by low energy electrons: A modelling approach

Abstract

Purpose: We propose a biochemical mathematical model for the repair of double-strand breaks (DSB) induced by low energy electron tracks, and determine the repair time for simple and complex DSB.

Materials and methods: The track structure code KURBUC_liq was used to simulate electron tracks in liquid water. All possible sites of energy depositions and reactions of water radicals in the nucleobases of an atomistic model of DNA were located, and the types of damage were determined. The initial induced DSB were subjected to a mechanistic model of Nonhomologous end-joining (NHEJ) repair.

Results: Data are presented for the initial and residual yield of DSB induced by low energy electrons. The model of repair was verified by comparing the kinetics of the unrejoined DSB with the experimental data for the V79?-?4 hamster cells irradiated with 15 Gy of Carbon-K (C_K) 278 eV ultrasoft X-rays. The residual unrepaired DSB in the duplex DNA is presented in the time interval up to 3 hours. The calculated repair time for the simple and complex DSB are presented.

Conclusion: With the hypothesis that complex DSB take longer time to repair than the simple type DSB, the model provides an estimate of DSB repair kinetics of experimental data.     

Differential response to acute low dose radiation in primary and immortalized endothelial cells

Abstract

Purpose: The low dose radiation response of primary human umbilical vein endothelial cells (HUVEC) and its immortalized derivative, the EA.hy926 cell line, was evaluated and compared.

Material and methods: DNA damage and repair, cell cycle progression, apoptosis and cellular morphology in HUVEC and EA.hy926 were evaluated after exposure to low (0.05–0.5 Gy) and high doses (2 and 5 Gy) of acute X-rays.

Results: Subtle, but significant increases in DNA double-strand breaks (DSB) were observed in HUVEC and EA.hy926 30 min after low dose irradiation (0.05 Gy). Compared to high dose irradiation (2 Gy), relatively more DSB/Gy were formed after low dose irradiation. Also, we observed a dose-dependent increase in apoptotic cells, down to 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells. Furthermore, radiation induced significantly more apoptosis in EA.hy926 compared to HUVEC.

Conclusions: We demonstrated for the first time that acute low doses of X-rays induce DNA damage and apoptosis in endothelial cells. Our results point to a non-linear dose-response relationship for DSB formation in endothelial cells. Furthermore, the observed difference in radiation-induced apoptosis points to a higher radiosensitivity of EA.hy926 compared to HUVEC, which should be taken into account when using these cells as models for studying the endothelium radiation response.     

Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

Abstract

Purpose: Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of targeted genes was examined.

Materials and methods: TFO were labeled with I-125 using the primer extension method. DNA triplex formation and sequence-specific DSB were demonstrated in vitro. Cell survival was analyzed by colony-forming assay and DNA damage was assessed by microscopic quantification of protein 53 binding protein 1 (53BP1) foci in the human squamous carcinoma cell line II (SCL-II). Quantitative real-time polymerase-chain-reaction (qRT-PCR) was performed to analyze gene expression alterations.

Results: The sequence-specific induction of a single DSB in a 1695 bp long DNA double stranded fragment was demonstrated in vitro. I-125-labeled TFO binding to single and multiple targets were shown to induce a pronounced decrease in cell survival and an increase of DSB. TFO targeting multiple sites differing in the total target number showed a significant different cell killing per decay that is also in good accordance with the observed induction of DSB. Single gene targeting I-125-labeled TFO significantly decreased cell survival and altered gene expression in the targeted gene.

Conclusions: I-125-labeled TFO enable specific targeting of DNA in vitro as well as in a cellular environment and thus induce sequence-specific complex DNA lesions. Therefore I-125-labeled TFO might be a very useful tool for basic DNA repair research.     

Cytotoxicity of cigarette smoke condensate is not due to DNA double strand breaks: Comparative studies using radiosensitive mutant and wild-type CHO cells

Purpose:?To determine whether cigarette smoke condensate (CSC) without metabolic activation induces direct DNA double strand breaks (DSB) in the G1 phase of various radiosensitive mutants of CHO cells and whether these breaks display collateral hypersensitivity to CSC with respect to cell killing.

Materials & methods:?We treated the G1-phase cultures of wild-type and DNA repair deficient mutants of CHO cells with various concentrations of CSC and examined the cell survival by colony formation assay and the induction of DNA double strand breaks by constant field gel electrophoresis as well as the phophorylated histone H2-A variant X (γ-H2AX) assay.

Results:?Gel analysis and γ-H2AX focus assay showed significantly fewer, but still detectable levels of DSB per cell after CSC treatment compared to ionizing radiation (IR) exposures, even when equitoxic radiation exposures were delivered at a low dose rate over the same 8-hour exposure used for CSC treatments. None of the three non-homologous end joining (NHEJ) deficient mutants were remarkably hypersensitive to CSC compared to wild-type cells. In contrast, UV-1 cells that are hypersensitive to several base damage and cross-linking agents showed a higher sensitivity to CSC compared to the other CHO cell lines.

Conclusions:?DNA DSB produced directly by CSC are not principally responsible for its cytotoxicity. Further, the present study does not rule out the possibility that some of these lesions may secondarily result in DSB, such as may occur during impeded DNA replication and whose repair may require systems other than NHEJ.     

Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?

PURPOSE: To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis. MATERIALS AND METHODS: The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Bl?cher model can be used to express data as DSB after irradiation at 4 degrees C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4 degrees C followed by incubation at 37 degrees C) in untransformed human fibroblasts. RESULTS: The DSB induction rate after irradiation at 4 degrees C was 39.1+/-2.0 Gy(-1). The DSB repair rate obtained after doses of 10 to 80 Gy followed by repair times of 0 to 24 h was expressed as unrepaired DSB calculated from the Bl?cher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar. CONCLUSION: The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Bl?cher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.     

DNA strand breakage by bivalent metal ions and ionizing radiation

Purpose: To investigate mechanisms of DNA breakage via the interaction of bivalent metal ion, thiol reducing agent and ionizing radiation, in ?OH scavenging abilities comparable to those in cells.

Materials and methods: We measured the effects of 10 min exposure to 200 μM Fe²⁺ vs. Fe³⁺ on the induction of single (SSB) and double (DSB) strand breaks in unirradiated and oxically irradiated SV40 DNA, in aqueous solution containing 75 or 750 mM glycerol and/or 5 mM glutathione (GSH).

Results: Fe²⁺ or GSH alone produced little DNA damage. However, their combination produced a dramatic increase in the production of both SSB and DSB. Experiments with ferric ion suggest that it produces DNA damage only after partial reduction to ferrous by GSH. Induction efficiencies for SSB in the presence of Fe²⁺/GSH showed additivity of the effects of radiation alone with those from Fe²⁺/GSH. However, the corresponding induction efficiencies for DSB demonstrated a 2.5-fold enhancement.

Conclusions: Our results are consistent with a model in which reduced bivalent metal ions plus thiols, in the presence of O₂, produce DSB in DNA primarily via local clusters of hydroxyl radicals arising from site specific Fenton reactions. The synergism observed between DSB production by Fe/GSH and by ionizing radiation, also believed to occur via local clusters of hydroxyl radicals, is consistent with this model. Our results suggest that both normally present intracellular iron and ionizing radiation may be important sources of oxidative stress in cells.