Elucidation of recognition mechanisms of antibodies specific for nucleic aicd derivatives

由紫外线（UV）光、烷化剂、放射性衰变或活性氧诱导的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。在DNA中相邻的吡啶位点上，形成三种主要的紫外光诱导的光产物，即顺式-顺环丁烷嘧啶二聚体（CPD）、嘧啶（6 - 4）嘧啶酮光产物[（6 - 4）光产物]和杜瓦异构体。单克隆抗体已被用作检测和定量这些DNA光损伤的手段。我们一直在研究一系列特异性识别CPD或（6 - 4）光产物的单克隆抗体，为了了解抗体的分子识别，我们克隆并测序了可变区基因。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。计算机模拟显示 ...更多信息 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。化学合成含有CPD或（6 - 4）光产物作为抗原的寡核苷酸。这些抗体的单链Fv（scFv）衍生物被构建和表征以提供可以测试结构-功能关系的实验系统。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。对（6 - 4）光产物特异性的scFv（64M5scFv）的互补决定区（CDR）的诱变研究揭示了在64M5抗体与DNA光产物结合中起重要作用的几个氨基酸残基。已经通过使用X射线晶体学分析了抗体64 M2、64 M3和64 M5的Fab片段的三维结构。在这些Fab中，6-4 M2 Fab作为与T（6 - 4）T配体复合的形式获得，并且清楚地阐明了结合的配体的结构以及配体识别模式。UV损伤的DNA与抗体64 M3和64 M5的Fab片段之间的相互作用的多核NMR研究显示了这些抗体的抗原结合位点和动态结合机制的结构信息。少

DNA damage induced by ultraviolet (UV) light, alkylating agents, radioactive decay or reactive oxygen is considered to be a principal cause of mutation, neoplastic cellular transformation and cell death. At adjacent pyridine sites in DNA, three major classes of UV-induced photoproducts, i.e. cis-syncyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] and the Dewar isomers are formed. Monoclonal antibodies have been used as a means to detect and quantitative these DNA photolesions. We have been studying a series of monoclonal antibodies that specifically recognize either CPDs or (6-4) photoproducts.In order to understand the molecular recognition of the antibodies, we have cloned and sequenced the variable region genes. Comparing these sequences revealed that all four anti-(6-4) photoproduct antibodies were highly similar to one another, although there were some differences in potential antigen-contact regions. Computer modeling have shown … More that 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. Oligonucleotides containing either CPDs or (6-4) photoproducts as the antigens were chemically synthesized. Single-chain Fv (scFv) derivatives of these antibodies were 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. Mutagenesis studies of complementarity-determining regions (CDRs) of scFv specific for (6-4) photoproduct (64M5scFv) revealed several amino acid residues that play an important role in DNA photoproduct binding by 64M5 antibody. Three-dimensional structures of the Fab fragments for antibodies 64M2, 64M3 and 64M5 have been analyzed by use of X-ray crystallography. Among these Fabs, 64M2 Fab was obtained as a form complexed with a T(6-4)T ligand and clearly elucidated the structure of the bound ligand as well as the ligand recognition mode. Multinuclear NMR studies of interactions between UV-damaged DNA and the Fab fragments for antibodies 64M3 and 64M5 have shown the structural information on the antigen binding sites and dynamic binding mechanisms of these antibodies. Less