DMA Lesions on the Specific Site in the Transcription Factor Binding Domain with RecA-Oligonucleotide Complexes

5-甲酰基-2 '-脱氧尿苷（fdU）是一种氧化性胸苷损伤，已知其在体内具有致突变性和细胞毒性。而这些unfavolable的生物学效应似乎在很大程度上是由复制和转录过程中的错配和代谢酶的抑制引起的，这是有趣的，考虑的反应，fU的甲酰基与蛋白质提供交联和它的生物学效应的影响。我们发现，含有5-甲酰基-2 '-脱氧尿苷（dfU）的单链寡核苷酸可以通过Schiff碱形成交联来自RecA蛋白的DNA结合位点的肽，从残基193延伸到212。使用一系列肽研究含fdU的寡核苷酸的交联能力，所述肽的跨越RecA衍生肽的中心的氨基酸残基依次被赖氨酸取代。圆二色谱（CD）和凝胶迁移率变动分析表明，只有当肽与寡核苷酸结合时，交联反应才能有效进行。沉降实验表明，交联产物呈聚集状态。这些结果表明，含fdU的寡核苷酸将是一个有用的工具，用于识别赖氨酸在蛋白质-DNA复合物中的核碱基附近。RecA蛋白可能是一种有用的载体，将修饰的核苷酸转运到目的基因。我们还研究了NFκB B和维生素D受体的其他转录因子。

5-Formy-2'-deoxyluidine (fdU) is an oxidative thymidine lesion, which has been known to be mutagenic and cytotoxic in vivo. Whereas these unfavolable biological effects of fU appear to be largely caused by mispairing during replication and transcription and inhibition of metabolic enzymes, it is intriguing to consider the reaction of formyl group of fU with proteins affording cross-link and its implications for the biological effects. We showed that single-stranded oligonucleotides containing 5-formyl-2'-deoxyuridine (dfU) can cross-link the peptides derived from the DNA binding site of RecA protein, extending from residues 193 to 212, through a Schiff base formation. The ability of cross-linking of fdU-containing oligonucleotides was investigated using a series of peptides whose amino acid residues spanning the center of the RecA-derived peptide were sequentially replaced with lysine. Circular dichroism (CD) spectroscopy and gel mobility shift assay demonstrated that cross-linking reaction proceeded effici ntly only when the peptides bound to the oligonucleotides. Furthermore, sedimentation experiment revealed that the final cross-linked products were in state of aggregation. These results demonstrate that fdU-containing oligonucleotides would be a useful tool for identifying lysines in close proximity to the nucleobase in protein-DNA complexes. RecA protein could be a useful carrier in transporting modified nucleotides to the target gene. We also investigated the other transcription factors of NFκB and vitamin D receptor.