激光作用质粒DNA和小牛胸腺DNA的AFM研究

激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ产生损伤效应,导致ＤＮＡ结构变化,利用一种改进的试样制备过程和纳米显微镜--原子力显微镜（ＡＦＭ）能够获得可重现的激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ的ＡＦＭ图象,显示它们的特殊的表面结构。     

补骨脂素—UVA对端粒DNA的直接作用及其原子力显微镜研究

HeLa细胞DNA,经Rsal(1250U)和HinF1(1250U)内切酶酶解、Bio-gel P-2柱分离,得到端粒DNA。原子力显微镜（AFM）的直接观测结果表明在UVA照射下,补骨脂素可结合到端粒DNA的特殊位置,其光交链产物随照射时间增长而增加。与此同时,P53表达和端粒酶活性发生变化。纯化的端凿DNA片段在AFM下呈带尾的T－环结构（T-loop）。按其高度判断T－环部分为双链DNA结构,T－环与尾部交接处具有三链或四链结构特征。     

小牛胸腺脱氧核糖核酸的提取和分析

在许多生化工作中,需要一定纯度和保持某些天然性质的脱氧核糖核酸(DNA)。鉴于这个目的,我们以小牛胸腺为材料,对DNA提取和纯化进行了多种方法的摸索,并对产品进行了初步鉴定。实验结果说明,按照确定的方法所获得的DNA符合一般生化工作的要求,这套方法也可供提取其它组织DNA时参考。方法 1.DNA的提取 (1)小牛胸腺染色质的分离详细过程见《小牛胸腺染色质组成分析》一文。 (2)核组蛋白的制备从100克小牛胸腺制备的染色质溶解在1,000毫升1M NaCl溶液     

抗癌药物奥沙利铂与DNA分子相互作用的研究

奥沙利铂被称为第三代铂类药物,特别对胃肠道肿瘤具有较好的疗效.目前大多数的研究表明奥沙利铂的主要作用靶点是DNA分子,但它与DNA分子形成的关键结构和作用机制仍处在探索阶段.本研究运用紫外可见吸收光谱和原子力显微镜观察探索奥沙利铂与DNA在活体外的相互作用过程,从而揭示奥沙利铂产生抗癌作用的主要分子结构基础.首先使用紫外光谱研究了较高浓度奥沙利铂与DNA的作用过程.在此基础上,进一步采用原子力显微镜在高定向热解石墨表面观察了不同浓度奥沙利铂与质粒DNA在37℃条件下作用不同时间后的结构形貌变化,分析了奥沙利铂与DNA相互作用的过程.高分辨原子力显微观察结果表明奥沙利铂与DNA作用后可导致质粒DNA的结构发生显著的变化.随着作用时间的增加,DNA分子逐渐由伸展的链状变化为相互缠绕并带有许多结点的紧密结构,最终变化为更紧密的球状结构.本研究结果表明奥沙利铂可通过化学键合作用和静电作用使质粒DNA逐渐凝集为紧密的球状结构,这种结构可能对奥沙利铂的抗癌活性和毒性产生重要影响.     

XeCl 准分子激光辐照生物大分子对其结构影响初步研究-Ⅰ308 nm紫外激光辐照小牛胸腺DNA的电镜观察与分析

XeCl 308 nm紫外激光(单脉冲输出能量33.4 mJ,脉冲频率 2次/秒,辐照时间45秒,光斑6×3 mm,透镜焦距300 mm)辐照330 mm处小牛胸腺DNA(固体).用扫描电子显微镜和透射电子显微镜可以观察到DNA受照射后有断裂、分叉、扭曲、聚集和交联等现象,影响生物大分子的初级结构和高级结构的变化.在我们的实验条件下是以影响DNA构象变化为主.     

DNA双链区的切割导致碱变性超螺旋质粒pBR322的分子内结节

为进一步研究碱变性超螺旋DNA(Ⅳ型DNA;DNA Ⅳ)的结构,对碱变性质粒pBR322进行了酶学分析并利用原子力显微镜(AFM)对新形成的DNA结构进行了观察和比较．在所有已检测的限制酶中只有PstⅠ可以切割质粒pBR322的Ⅳ型DNA分子,说明在这种变性的DNA分子中仍存在少数完整的限制酶识别位点．与碱变性DNA分子的闭合环状结构相反,AFM成像的结果显示PstⅠ处理后的DNA Ⅳ分子均为开放结构,同时这种分子包含明显的DNA结节．与DNA Ⅳ分子相比,这种DNA分子的表观长度缩短了大约11%．有意思的是,大肠杆菌拓扑异构酶Ⅳ(一种Ⅱ型拓扑异构酶)也可以在pBR322 DNA Ⅳ分子中引入分子内结节,而这种结节DNA分子仍然保持闭合状态．AFM的结果表明上述两种结节的DNA分子在表观长度及结节结构的尺寸上均比较相似．这些发现证实,在碱变性的超螺旋DNA分子中仍然保留着一些长度较短的、含有特异性碱基配对的DNA双链区,而在这些区域内的DNA双链断裂可以导致DNA结节．     

4′6二脒基-2-苯基吲哚与DNA结合特性研究

本文以小牛胸腺DNA和λDNA为材料,首次用激光拉曼光谱研究了4’6二脒基—2—苯基吲哚(DAPI)与DNA的结合机制.认为DAPI是以非镶嵌的方式在DNA小沟中同DNA的AT碱基对此氢键结合.在低PH下,这种结合受到抑制,盐浓度的高低与DAPI-DNA复合体的荧光强度成负相关.     

4′6二脒基-2-苯基吲哚与DNA结合特性研究

本文以小牛胸腺DNA和λDNA为材料,首次用激光拉曼光谱研究了4’6二脒基—2—苯基吲哚(DAPI)与DNA的结合机制.认为DAPI是以非镶嵌的方式在DNA小沟中同DNA的AT碱基对此氢键结合.在低PH下,这种结合受到抑制,盐浓度的高低与DAPI-DNA复合体的荧光强度成负相关.     

DNA单分子近场光学成像与荧光探测

介绍了扫描近场光学(SNOM-Scanning Near-Field Optical Microscope)／原子力显微镜（AFM－Atomic Force Microscope)系统（SNO／AM）的工作原理。在AFM模式和SNOM模式下对DNA分子进行成像和荧光探测,得到了清晰的DNA单分子的形貌像和荧光像。由形貌圆像得到的DNA分子尺寸横向为20nm,高度为2nm,其中包含了探针形貌的影响。实验中采Tapping模式的AFM成像,样品经多次搜索扫描无明显损坏。AFM模式的分辨率优于1nm。SNOM模式下DNA分子形貌像和荧光像清晰,由近场荧光分布可以确定分子取向和浓度。用YOYO－1染料对λDNA分子进行染色和荧光探测。通过对DNA分子多个截面进行测量,分析染料 与DNA结合状态。     

姜黄素与DNA相互作用的电化学研究

目的:利用电化学方法研究姜黄素与小牛胸腺双链DNA的相互作用,考察离子强度对姜黄素与DNA相互作用方式的影响.探讨姜黄素与DNA相互作用的机理.方法:在5 mmol·L-1 Tris-HCl(pH 7.0)溶液中,在不同离子强度下,考察姜黄素在ds-DNA修饰玻碳电极上的姜黄素氧化峰电流及式电位的变化情况.结果:在低离子强度下,姜黄素主要通过静电作用与DNA结合.高离子强度下,姜黄素主要通过嵌入作用与DNA结合.结论:电化学方法是研究姜黄素与电极表面固定化小牛胸腺双链DNA的相互作用的一种简单的方法.     

激光对DNA的作用

简述了激光对DNA的作用。介绍了与激光辐照导致DNA链断裂、光产物形成及其分子机理、激光损伤与突变的关系以及高能脉冲激光和双光子作用有关的一些研究结果。     

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。     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

The recovery parameters of DNA strand breaks of Ehrlich ascites cells after the combined action of ionizing radiation and hyperthermia

A mathematical model of DNA strand breaks postirradiation repair and the methodology allowing to differentiate the mechanism of inhibition of DNA strand breaks recovery after combined actions of ionizing radiation and hyperthermia have been described in this paper. Using this model and the results published by other authors for DNA strand breaks of Ehrlich ascites cells, there have been obtained the data showing that the portion of DNA-damages that the cell incapable to recover after consecutive thermoradiation action was risen with an increase in thermal load under insignificant change of repair constant. It means the mechanism of DNA strand breaks recovery inhibition is realized in a greater extent through the formation of irreversible damages but not through the damage of repair process itself.     

PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.     

Attempted base excision repair of ionizing radiation damage in human lymphoblastoid cells produces lethal and mutagenic double strand breaks

A significant proportion of cellular DNA damages induced by ionizing radiation are produced in clusters, also called multiply damaged sites. It has been demonstrated by in vitro studies and in bacteria that clustered damage sites can be converted to lethal double strand breaks by oxidative DNA glycosylases during attempted base excision repair. To determine whether DNA glycosylases could produce double strand breaks at radiation-induced clustered damages in human cells, stably transformed human lymphoblastoid TK6 cells that inducibly overexpress the oxidative DNA glycosylases/AP lyases, hNTH1 and hOGG1, were assessed for their radiation responses, including survival, mutation induction and the enzymatic production of double strand breaks post-irradiation. We found that additional double strand breaks were generated during post-irradiation incubation in uninduced TK6 control cells. Moreover, overproduction of either DNA glycosylase resulted in significantly increased double strand break formation, which correlated with an elevated sensitivity to the cytotoxic and mutagenic effects of ionizing radiation. These data show that attempted repair of radiation damage, presumably at clustered damage sites, by the oxidative DNA glycosylases can lead to the formation of potentially lethal and mutagenic double strand breaks in human cells.     

Caged single and double strand breaks

Ionizing radiation and radiomimetic drugs such as bleomycin, calichieamycin, neocarzinostatin chromophore, and other synthetic agents can produce both single and double strand breaks in DNA. The ability to study the structure-activity relationships of single and double-strand break repair, lethality, and mutagenesis in vivo is complicated by the numerous types and sites of DNA cleavage products that can be induced by such agents. The ability to "cage" such breaks in DNA might help to further such studies and additionally afford a mechanism for activating and deactivating nucleic acid based drugs and probes. The major type of single strand break induced by ionizing radiation is a 3'- and 5'-phosphate terminated single nucleotide gap. Previously, a caged strand break of this type had been developed that was designed to produce the 5'-phosphate directly upon irradiation with 366 nm light, and the 3'-phosphate by a subsequent beta-elimination reaction [Ordoukhanian, P., and Taylor, J.-S. (1995) J. Am. Chem. Soc. 117, 9570]. Unfortunately, the release of the 3'-phosphate group was quite slow at pH 7. To circumvent this problem, a second caged strand break has been developed that produces the 3'-phosphate directly upon irradiation, and the 5'-phosphate by a subsequent beta-elimination reaction. When this caged strand break was used in tandem with the previous caged strand break, 5'- and 3'-phosphate terminated gaps could be directly produced by irradiation with 366 nm light. These caged single strand breaks were also incorporated in tandem into hairpin substrates to demonstrate that they could be used to cage double strand breaks. These caged single strand breaks should be generally useful for generating site-specific DNA single and double strand breaks and gaps, using wavelengths and doses of light that are nondetrimental to biological systems. Because the position of the single strand break can be varied, it should now be possible to examine the effect of the sequence context and cleavage pattern of single and double strand breaks on the lethality and mutagenicity of this important class of DNA damage.     

Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.     

Quantitative assessment of the contribution of clustered damage to DNA double-strand breaks induced by 60Co gamma rays and fission neutrons

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.