抗辐射菌中DNA损伤修复主要基因群的研究进展

抗辐射红色球菌对电离辐射具有很高的放射线抵抗性,该菌具有惊人的DNA的二条链切断的修复能力,由辐射等引起的切断损伤DNA在几至十几小时内能高效正确地进行完全修复。在对切断的双链DNA进行修复时,除了大肠杆菌等生物在切断的双链DNA修复时出现的蛋白质以外,还有该菌所特有的修复蛋白质也参与修复。本文对该菌所特有的DNA二条链的切断损伤修复的主要基因及其相互作用进行了简要介绍。     

重离子射线照射对家蚕的生物影响

为解明重离子射线的生物影响,调查了氖、碳及氦(20Ne8+,LET=300keV/μm;12C5+,LET=116keV/μm和4He2+,LET=16.2keV/μm)等重离子射线照射家蚕(Bombyxmori)后的存活率及形态变化。重离子射线照射不同发育时期的幼虫后所引起的生物影响不同,幼虫的发育时期越早,照射后引起的生物影响越大;对同一时期的幼虫,随着剂量的增加,照射的生物影响加大;以化蛹率和羽化率为指标的放射线感受性在供试的3种射线间具有相似的变化倾向,只是射线的射程越长,照射的生物影响越大;对熟蚕卵巢存在部位的局部照射也显示相似的结果。同一射线的不同LET轨迹位置对家蚕的卵巢及真皮细胞的生物影响不同,用Mylar薄膜覆盖调节碳离子射线的射程,卵巢及真皮细胞越是接近射线高LET的Bragg峰,照射个体的鳞毛及卵的形成被强烈抑制。因此,重离子射线对家蚕的生物影响与细胞及植物种子等小个体不同,对于全体照射,重离子射线的射程长短所造成的生物影响比射线的LET大小所引起的生物影响要大;而对于局部照射,目的器官越是接近射线的高LET轨迹,照射的生物影响越大。     

不同LET的重离子辐射对DSB诱导的影响

利用γ射线和不同LET的碳离子辐照小鼠B16黑色素瘤细胞的脱蛋白DNA,采用脉冲场凝胶电泳结合荧光扫描技术研究了DNA双链断裂（DSB）与LET之间的关系。结果表明：不同LET重离子诱导的PR都随剂量的增加而增加,并在超过一定的剂量之后逐渐趋于一个准阈值;而不同LET的重离子诱导的L值都与剂量呈线性关系;对于诱导DSB的RBE值则随着LET的增加先呈上升趋势,在LET超过100ke/μm后下降。     

80MeV/u20Ne10+离子对SMMC-7721细胞DNA损伤及细胞周期的影响

从辐照剂量和修复时间两个角度研究了重离子辐照对肿瘤细胞DNA损伤及细胞周期的影响,为重离子治癌的临床应用积累基础数据。不同剂量的80MeV／u^20Ne^10 辐照SMMC—7721细胞样品,利用单细胞凝胶电泳技术(Single Cell Gel Electrophoresis,SCGE)对细胞DNA损伤进行了检测,利用流式细胞技术(Flow Cytometry Methods,FCM)对细胞周期变化进行了分析。80MeV／u^20Ne^10 辐照后4小时内,SMMC—7721细胞的DNA损伤与辐照剂量呈线性关系,在0小时组其线性相关因子r为0．9621,4小时组为0．914;随着修复时间的增加,DNA损伤与辐照剂量不再线性相关,但0．5Gy,1Gy和2Gy三个剂量点的DNA损伤程度极为相近。另外,重离子辐照后SMMC—7721细胞发生S期和G2／M期阻滞现象,其随剂量变化及时间变化的规律不同于X、γ等低LET(Linear Energy Transfer)射线辐照。     

单能质子辐射致质粒DNA链断裂的剂量效应

利用16.4 MeV的质子在不同的剂量下辐照质粒DNApUC19溶液。凝胶电泳技术的分析结果表明随着辐照剂量的增加,DNA损伤变得越来越严重,使线性DNA成分明显增加。当添加了自由基清除剂甘露醇后,DNA的损伤明显减轻,线性DNA片段不再出现,但开环形态DNA的变化依然明显。与较早的重离子7Li和γ射线致DNA损伤的研究结果相比较,表明质子辐射中还是存在着一定的直接作用,在此次实验的能量和LET值范围内,质子的直接电离作用是高于γ射线而低于7Li离子的。     

单细胞凝胶电泳联合原子力显微镜观测UVB诱导的细胞DNA损伤模式变化

目的：检测UVB诱导的真核细胞DNA损伤。方法：采用单细胞凝胶电泳与原子力显微镜。结果：不同照射剂量的UVB引起的真核细胞DNA损伤模式不同。在0～20J／m2照射剂量范围内DNA无损伤;在20--360J／m2照射剂量范围内DNA损伤程度加快;当照射剂量超过360J／m2时DNA损伤速度减慢,实验组之间无显著性差异,出现“平台”。原子力显微镜的观察结果表明随着UVB照射剂量的增加,DNA结构的变化经历了断裂、交联与断裂并存的损伤增强趋势。当照射能量达到280J／m2时细胞DNA大都形成断片,并相互交联在一起。这一结果表明彗星电泳检测到的UVB照射剂量达到一定剂量后,DNA损伤出现”平台”的原因可能是此时DNA发生了链内或链间交联。结论：不同照射剂量的UVB造成的细胞DNA损伤模式不同;原子力显微镜是一种比较直观的观测DNA损伤的方法。借助原子力显微镜我们可以深入了解单细胞凝胶电泳检测的原理,为DNA损伤检测提供更优良的检测手段。     

抗辐射菌Deinococcus radiodurans的多样性

抗辐射菌Deinococcus radiodurans是一种对电离辐射和其他DNA损伤因子具有极强抵抗能力的细菌,是研究DNA损伤与修复的模式生物。综述了国内外在抗辐射菌研究上取得的最新研究成果,从生存环境、对DNA损伤因子的抗性、抗性机理及其损伤修复关联基因等方面报道了抗辐射菌的多样性,并探讨了该细菌高效正确的DNA损伤修复机理的相关研究成果在生命科学、农业、环境修复及医学等领域的应用前景。     

损伤DNA 的“SOS”修复

活体细胞内的DNA会受到许多外界因素,诸如射线、带电离子、化学药物等的损伤。在活细胞中,有一系列原有的或由损伤因素诱导产生的酶,能对DNA的各种损伤进行不同形式的修复。易错修复(error-prone DNA repair),即修复时容易发生错误。它是M.Radman首先发现的。他用紫外线照射λ噬菌体和ΦX174噬菌体,然后用这种噬菌体去感染经紫外线轻度照射过的大肠杆菌,结果是被照射过的λ噬菌     

重离子的辐射生物效应及其在生命科学中的应用

重离子是指重于元素周期表中2号元素氦并被电离的粒子。高能重离子因在其穿透物质的径迹上产生很强的局部电离,与传统的光子辐射(如X、γ射线)相比,会诱导更严重的辐射损伤生物效应。目前的机理研究显示,重离子辐射造成细胞DNA局部一到两个螺旋内出现多类型、多数量的损伤,即DNA团簇损伤。这种复杂的损伤影响DNA修复酶与DNA片段结合,进而影响修复,导致细胞死亡或产生不正确修复(即突变)。重离子辐射易引起突变的特征在植物与微生物诱变育种方面得到青睐。此外,重离子的细胞杀伤作用大,而且重离子具有倒转的剂量分布优势,将其用于癌症治疗,可使深部区域肿瘤细胞因高剂量辐射而被有效杀灭,而对周围组织因低剂量分布、相对危害较小,能实现精确靶向治疗,这被认为是最有前景的肿瘤放疗技术。对由重离子造成的DNA团簇损伤及其修复机制,以及两个重要修复途径的研究进展,进行了总结与深入分析。此外,我们还对重离子在辐射诱变育种和肿瘤治疗方面的应用进行了概要介绍,以期为从事重离子辐射生物学研究的人员理解DNA团簇损伤与修复机制及未来研究方向提供参考,促进对重离子辐射危害的评估与防护策略的建立、及其在生命科学中的良好应用。     

辐射及活性氧对DNA的损伤以及芥子碱的保护作用

在X射线照射下,小牛胸腺DNA的碱基损伤及链断裂随着剂量升高而增加,其损伤主要集中于链断裂;活性氧可以引起DNA损伤,H2O2仅造成少量伤害,当在含有H2O2的体系中加入微量的Cu2＋、Fe2＋时损伤急剧增加,这是由反应产生的·OH所致,Cu2＋的致损伤效果明显高于Fe2＋。·OH清除剂芥子碱具有很强的抗辐射及抗氧化作用,且对DNA无伤害。这说明·OH在DNA的氧化损伤中起重要作用。     

The complexity of DNA double strand breaks is a critical factor enhancing end-resection

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) are deleterious damages. Two major pathways repair DSBs in human cells, DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been suggested that the balance between the two repair pathways varies depending on the chromatin structure surrounding the damage site and/or the complexity of damage at the DNA break ends. Heavy ion radiation is known to induce complex-type DSBs, and the efficiency of NHEJ in repairing these DSBs was shown to be diminished. Taking advantage of the ability of high linear energy transfer (LET) radiation to produce complex DSBs effectively, we investigated how the complexity of DSB end structure influences DNA damage responses. An early step in HR is the generation of 3′-single strand DNA (SSD) via a process of DNA end resection that requires CtIP. To assess this process, we analyzed the level of phosphorylated CtIP, as well as RPA phosphorylation and focus formation, which occur on the exposed SSD. We show that complex DSBs efficiently activate DNA end resection. After heavy ion beam irradiation, resection signals appear both in the vicinity of heterochromatic areas, which is also observed after X-irradiation, and additionally in euchromatic areas. Consequently, ∼85% of complex DSBs are subjected to resection in heavy ion particle tracks. Furthermore, around 20–40% of G1 cells exhibit resection signals. Taken together, our observations reveal that the complexity of DSB ends is a critical factor regulating the choice of DSB repair pathway and drastically alters the balance toward resection-mediated rejoining. As demonstrated here, studies on DNA damage responses induced by heavy ion radiation provide an important tool to shed light on mechanisms regulating DNA end resection.     

Differences in Phosphorylated Histone H2AX Foci Formation and Removal of Cells Exposed to Low and High Linear Energy Transfer Radiation

The use of particle ion beams in cancer radiotherapy has a long history. Today, beams of protons or heavy ions, predominantly carbon ions, can be accelerated to precisely calculated energies which can be accurately targeted to tumors. This particle therapy works by damaging the DNA of tissue cells, ultimately causing their death. Among the different types of DNA lesions, the formation of DNA double strand breaks is considered to be the most relevant of deleterious damages of ionizing radiation in cells. It is well-known that the extremely large localized energy deposition can lead to complex types of DNA double strand breaks. These effects can lead to cell death, mutations, genomic instability, or carcinogenesis. Complex double strand breaks can increase the probability of mis-rejoining by NHEJ. As a consequence differences in the repair kinetics following high and low LET irradiation qualities are attributed mainly to quantitative differences in their contributions of the fast and slow repair component. In general, there is a higher contribution of the slow component of DNA double strand repair after exposure to high LET radiation, which is thought to reflect the increased amount of complex DNA double strand breaks. These can be accurately measured by the γ-H2AX assay, because the number of phosphorylated H2AX foci correlates well with the number of double strand breaks induced by low or / and high LET radiation.     

Effect of heavy ion irradiation on DNA DSB repair in Methanosarcina barkeri

Archaea are expected to be highly repair proficient since they survived the vicious onslaught of radiation damage at the time of their early appearance. The DNA double strand break repairing ability of mesophilic archaea Methanosarcina barkeri (DSM 804) was studied using (7)Li, (12)C and (16)O heavy ions and compared with that of (60)Co gamma-rays. They can repair double strand breaks and, as in eukaryotes, the nature as well as extent of induction and its subsequent repair were dependent on the linear energy transfer of the radiation source.     

A track structure model for simulation of strand breaks in plasmid DNA after heavy ion irradiation

We present a track structure model based on the local dose deposited around heavy ion tracks to explain the cross sections for single-strand and double-strand break induction in plasmid DNA in different aqueous buffers. The model is based only on measurable quantities, namely the effect distribution for inducing strand breaks after x-ray irradiation as a function of dose, and the radial dose distribution of the heavy ion track. The effect of indirect DNA damage mediated by free radicals produced in the water surrounding the DNA is accounted for by allowing the radial dose distribution to be smeared in space by an effective target size corresponding to the squared sum of the geometrical extension of the plasmid molecule and the mean free drift path of the radicals in the buffer solution. Our calculations reproduce well the measured cross sections for single-strand and double-strand break induction in SV40 plasmid DNA in various buffer solutions both as a function of the LET and of the specific energy of the heavy ion.     

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2 Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.     

Clustered DNA damage induced by heavy ion particles.

Clustered DNA damage (locally multiply damaged site) is thought to be a critical lesion caused by ionizing radiation, and high LET radiation such as heavy ion particles is believed to produce high yields of such damage. Since heavy ion particles are major components of ionizing radiation in a space environment, it is important to clarify the chemical nature and biological consequences of clustered DNA damage and its relationship to the health effects of exposure to high LET particles in humans. The concept of clustered DNA damage emerged around 1980, but only recently has become the subject of experimental studies. In this article, we review methods used to detect clustered DNA damage, and the current status of our understanding of the chemical nature and repair of clustered DNA damage.     

DNA strand breaks based on Monte Carlo simulation in and around the Lipiodol with flattening filter and flattening filter-free photon beams

BackgroundThe current study aims to investigate the DNA strand breaks based on the Monte Carlo simulation within and around the Lipiodol with flattening filter (FF) and flattening filter-free (FFF) photon beams.Materials and methodsThe dose-mean lineal energy (y_D) and DNA single- and double strand breaks (DSB/SSB) based on spatial patterns of inelastic interactions were calculated using the Monte Carlo code: particle and heavy ion transport system (PHITS). The ratios of dose using standard radiation (200 kVX) to the dose of test radiation (FF and FFF of 6 MV X-ray (6MVX) and 10 MVX beams) to produce the same biological effects was defined as RBE_DSB. The RBE_DSB within the Lipiodol and in the build-up and build-down regions was evaluated.ResultsThe RBE_DSB values with the Lipiodol was larger than that without the Lipiodol at the depth of 4.9 cm by 4.2% and 2.5% for 6 MVX FFF and FF beams, and 3.3% and 2.5% for 10 MVX FFF and FF beams. The RBE_DSB values with the Lipiodol was larger than that without the Lipiodol at the depth of 6.5 cm by 2.9% and 2.4% for 6 MVX FFF and FF beams, and 1.9% and 1.4% for 10 MVX FFF and FF beams. In the build-down region at the depth of 8.1 cm, the RBE_DSB values with the Lipiodol was smaller than that without the Lipiodol by 4.2% and 2.9% for 6 MVX FFF and FF beams, and 1.4% and 0.1% for 10 MVX FFF and FF beams.ConclusionsThe current study simulated the DNA strand break except for the physical dose difference. The lower and FFF beam occurred the higher biological effect.     

High LET heavy ion radiation induces lower numbers of initial chromosome breaks with minimal repair than low LET radiation in normal human cells

We investigated the earliest possible chromosome break and repair process in normal human fibroblasts irradiated with low and high LET (linear energy transfer) heavy ion radiation using the modified premature chromosome condensation (PCC) technique utilizing wortmannin (WM) during the fusion incubation period [M. Okada, S. Saito, R. Okayasu, Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation, Mutat. Res., 562 (2004) 11-17]. The initial numbers of breaks were approximately 10/cell/Gy in X-irradiated samples, followed by carbon (LET: 70 keV/microm), neon, and the number was around 5/cell/Gy in silicon (LET: 70 and 200 keV/microm) and iron (LET: 200 keV/microm) samples. If WM was not used, the initial numbers of breaks with silicon and iron were higher than those of X-rays. To quantify these data, we used initial repair ratio (IRR) defined as the number of G1 PCC breaks with WM divided by the number of breaks without WM. X-irradiation gave the maximum IRR ( approximately 2.0), while iron as well as silicon irradiation showed the minimum IRR ( approximately 1.0), suggesting almost no rejoining at the initial stage. Although there is a comparatively good correlation between the IRR value and the cell survival, the survival fraction with the repair data at 2 or 6h correlates better statistically. Our data indicate that high LET heavy ion irradiation induces a lower number of initial chromosome breaks with minimal repair when compared with low LET irradiation. These results at the chromosome level substantiate and extend the notion that high LET radiation produces complex-type DNA double strand breaks (DSBs).     

A Stochastic Model of DNA Fragments Rejoining

When cells are exposed to ionizing radiation, DNA damages in the form of single strand breaks (SSBs), double strand breaks (DSBs), base damage or their combinations are frequent events. It is known that the complexity and severity of DNA damage depends on the quality of radiation, and the microscopic dose deposited in small segments of DNA, which is often related to the linear transfer energy (LET) of the radiation. Experimental studies have suggested that under the same dose, high LET radiation induces more small DNA fragments than low-LET radiation, which affects Ku efficiently binding with DNA end and might be a main reason for high-LET radiation induced RBE [1] since DNA DSB is a major cause for radiation-induced cell death. In this work, we proposed a mathematical model of DNA fragments rejoining according to non-homologous end joining (NHEJ) mechanism. By conducting Gillespie''s stochastic simulation, we found several factors that impact the efficiency of DNA fragments rejoining. Our results demonstrated that aberrant DNA damage repair can result predominantly from the occurrence of a spatial distribution of DSBs leading to short DNA fragments. Because of the low efficiency that short DNA fragments recruit repair protein and release the protein residue after fragments rejoining, Ku-dependent NHEJ is significantly interfered with short fragments. Overall, our work suggests that inhibiting the Ku-dependent NHEJ may significantly contribute to the increased efficiency for cell death and mutation observed for high LET radiation.     

不同离子辐照对离体质粒DNA损伤与转化活性的影响

利用 30Kev的N+ 、Ar+ 离子辐照离体质粒DNA ,分析了不同离子对DNA单双链断裂及转化活性的影响。结果表明 :N+ 、Ar+ 离子辐照均可引起质粒DNA单双链断裂和转化活性的变化 ,且随着辐照剂量的增加 ,单双链断裂频率增加 ,转化活性下降。Ar+对离体质粒DNA比N+ 具有更强的单双链断裂效应 ,且从 9× 10 15Ar+ cm2 剂量开始 ,质粒可完全丧失转化活性。质粒转化活性的大小与DNA单双链断裂频率呈正相关