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

RecA-寡核苷酸复合物对转录因子结合域特定位点的 DMA 损伤

• 批准号: 13672230
• 负责人: KITTAKA Atsushi
• 金额: $ 2.3万
• 依托单位: Teikyo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672230/
• 关键词: RecA Protein; gene recombination enzyme; oligonucleotides; Schiff base; NFκB; nuclear receptor; active vitamin D3; super agonist; CDスペクトル; ゲル電気泳動; 凝集; ホルミルウリジン

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.

5形-2'-脱氧酰胺（FDU）是一种氧化胸苷病变，已知在体内已知诱变和细胞毒性。尽管FU的这些不可捕获的生物学作用似乎主要是由于复制和转录过程中的错误引起的，并抑制了代谢酶的抑制作用，但考虑到甲基FU的反应与蛋白质的反应及其对生物学作用的影响及其对交叉链接的反应。我们表明，含有5-甲基-2'-脱氧尿苷（DFU）的单链寡核苷酸可以通过Schiff碱基形成从残留193到212的RECA蛋白的DNA结合位点衍生出的肽。使用一系列肽的氨基酸在横跨雷亚德肽的中心生存的氨基酸存活的一系列肽的交联能力被依次用赖氨酸代替。圆二色性（CD）光谱和凝胶迁移率转移测定表明，仅当彼得斯与寡核苷酸结合时，交联反应才有效进行。此外，沉积实验表明，最终的交联产物处于聚集状态。这些结果表明，含FDU的寡核苷酸将是一个有用的工具，可用于识别与蛋白质DNA复合物中核基础相近的歌词。 RECA蛋白可能是将修饰的核苷酸转运到靶基因的有用载体。我们还研究了NFκB和维生素D受体的其他转录因子。

项目成果

Toru Sugiyama, et al.: "Evidence of a Schiff Base Formation of Peptides Derived from RecA with Single-stranded Oligonucleotides Containing 5-Formyl-2'-deoxyuridine"Nucleic Acids Res.Suppl.. No.1. 175-176 (2001)

Toru Sugiyama 等人：“来自 RecA 的肽与含有 5-甲酰基-2-脱氧尿苷的单链寡核苷酸形成席夫碱的证据”，核酸研究补编第 1 号。

Yoshitomo Suhara, Atsushi Kittaka. Seishi Kishimoto, Martin J. Calverley, Toshie Fujishima, Nozomi Saito, Takayuki Sugiura, Keizo Waku, and Hiroaki Takayama: "Synthesis and Testing of 2α-Modified 1α,25-Dihydroxyvitamin D3 Analogues with a Double Side Chai

Yoshitomo Suhara、Atsushi Kittaka、Martin J. Calverley、Toshie Fujishima、Nozomi Saito、Takayuki Sugiura、Keizo Waku 和 Hiroaki Takayama：“具有双侧链的 2α-修饰 1α,25-二羟基维生素 D3 类似物的合成和测试”

Toru Sugiyama, et al.: "Chemical Cross-linking of Peptides Derived from RecA with Single-stranded Oligonucleotides Containing 5-Formyl-2'-deoxyuridine"Nucleosides Nucleotides & Nucleic Acids. 20巻・47号. 1079-1083 (2001)

Toru Sugiyama 等人：“RecA 衍生肽与含有 5-甲酰基-2-脱氧尿苷的单链寡核苷酸的化学交联”核苷核苷酸和核酸，第 20 卷，第 47 期。1079-1083 (2001)。

Atsushi Kittaka, et al.: "DNA Sequence Recognition by NaκB p50 Homodimer : Strict and Obscure Recognition Sites in the Binding Sequence"Nucleosides Nucleotides & Nucleic Acids. 20巻47号. 669-672 (2001)

Atsushi Kittaka 等人：“NaκB p50 同二聚体的 DNA 序列识别：结合序列中严格且模糊的识别位点”《核苷核苷酸与核酸》第 20 卷，第 47 期。669-672 (2001)。

高山 浩明: "活性型ビタミンD_3のA環修飾、特に2位の官能基化と生物活性"ビタミン. 76巻・11号. 491-505 (2002)

Hiroaki Takayama：“活性维生素 D_3 的 A 环修饰，特别是 2 位的功能化和生物活性”，维生素，第 76 卷，第 11 期，491-505 (2002)。