Recognition of DNA by thymine dimers specific antibodies

胸腺嘧啶二聚体特异性抗体对 DNA 的识别

• 批准号: 07044214
• 负责人: INOUE Hideo
• 金额: $ 6.53万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07044214/
• 关键词: UV-damaged DNA; monoclonal antibody; single-chain antibody; thymine dimer; (6-4) photo product; Dewar isomer; ELISA; Dewar型変性体

DNA damage induced by ultraviolet (UV) light is considered to be a principal cause of mutation, neoplastic cellualr transformation and cell death. The major classes of UV-induced photoproducts are cis-syn cyclobutane pyrimidine dimers (CPDs) , pyrimidine (6-4) pyrimidone photoproducts [ (6-4) photoproducts] and the Dewar isomers formed between adjacent pyrimidines in DNA.In order to detect and quantitate these DNA photoproducts, several monoclonal antibodies have been established. We have been studying a series of monoclonal antibodies that specifically recognize either CPDs or (6-4) photoproducts.In order to understand the molecular recognitions of the antibodies for the photoproducts, we have cloned and sequenced the variable region genes. Comparing these sequences revealed that all four anti- (6-4) photoproducts antibodies were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differen … More ces at the structural level, computer models were constructed for these antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. Single-chain Fv derivatives of these antibodies were therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. These derivatives could be isolated from E.coli using metal affinity chromatographic steps and possessed the same binding specificities as the parent monoclonal antibodies. The binding constants for octadeoxynucleotide containing either CPDs or (6-4) photoproducts as to both scFvs and corresponding Fabs were compared by means of BIAcore biosensor. The dissociation rate constants (kdiss) of scFv for the antigen analog was found to be almost the same as that of Fab, while the association rate constant (kass) of 2scFv was two-fold higher than that of Fab, These results indicate that the antigen binding region of the scFv seems to function as that of the intact antibody. Furthermore, elevated concentrations of NaCl decreased kass values of both Fab and scFv drastically, suggesting that these antibodies bind to oligonucleotide containing photoproducts through electrostatic interactions. Less

紫外线（UV）诱导的DNA损伤被认为是突变、肿瘤细胞转化和细胞死亡的主要原因。紫外光诱导的光产物主要有顺式环丁烷嘧啶二聚体（CPD）、嘧啶（6-4）嘧啶酮光产物[（6-4）光产物]以及DNA中相邻嘧啶之间形成的杜瓦异构体。为了检测和定量这些DNA光产物，已经建立了几种单克隆抗体。本实验室一直在研究一系列能特异性识别CPD或（6-4）光产物的单克隆抗体，为了了解这些抗体的分子生物学特性，我们克隆了抗体可变区基因并进行了序列测定。比较这些序列显示，所有四种抗-（6-4）光产物抗体彼此高度相似，尽管在潜在的抗原接触区域中存在一些差异。为了评估这些序列的影响， ...更多信息 在结构水平上，构建了这些抗体的计算机模型。大多数序列差异发生在潜在的抗原接触区域，表明可能解释所观察到的结合亲和力和选择性差异的特定位置。因此，这些抗体的单链Fv衍生物的构建和表征，以提供一个实验系统，其中结构-功能关系可以进行测试。这些衍生物可以使用金属亲和层析步骤从大肠杆菌中分离，并且具有与亲本单克隆抗体相同的结合特异性。通过BIAcore生物传感器比较了含有CPD或（6-4）光产物的八脱氧核苷酸与scFv和相应Fab的结合常数。结果表明，单链抗体对抗原类似物的解离速率常数（kdiss）与Fab几乎相同，而2scFv的结合速率常数（kass）是Fab的2倍，表明单链抗体的抗原结合区与完整抗体的抗原结合区具有相同的功能。此外，升高浓度的NaCl显著降低Fab和scFv两者的kass值，表明这些抗体通过静电相互作用与含有光产物的寡核苷酸结合。少

项目成果

Yasuo Komatsu.: "Antigen structural requirements for recognition by cyclobutane thymine dimer-specific monoclonal antibody." Nucleic Acids Res.25. 3889-3894 (1997)

Yasuo Komatsu：“环丁烷胸腺嘧啶二聚体特异性单克隆抗体识别的抗原结构要求。”

Takeshi Todo: "Flavin adenine dinucleotide as a choromophore of the Xenopus (6-4) photolyase" Nucleic Acids Res.25. 764-768 (1997)

Takeshi Todo：“黄素腺嘌呤二核苷酸作为非洲爪蟾 (6-4) 光裂合酶的发色团”Nucleic Acids Res.25。

Takeshi Toda: "Flavin adenine dinucleotide as a choromophore of the Xenopus(6-4)photolyase." Nucleic Acids Res.25. 764-768 (1997)

Takeshi Toda：“黄素腺嘌呤二核苷酸作为非洲爪蟾 (6-4) 光裂合酶的发色团。”