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標題: 以結構為基礎探討由棘黴素辨識含胸腺嘧啶錯配之序列
Structural basis of the specific binding of echinomycin to DNA sequence with T:T mismatch
作者: 高雅芬
Ya-Fen Kao
關鍵字: 棘黴素;T:T 錯配鹼基;DNA 解旋;Echinomycin;T:T mismatch;DNA unwinding
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In the process of cell division, the fidelity of the genetic information is ensured by proofreading of DNA polymerase and mismatch repair system. But there are still some mismatches occur. If the DNA repair system is disable to repair the mismatches, it will lead to the occurrence of genetic disease or cancer. It's very significant on human health. DNA intercalators have long been known as anticancer drugs, which have been well studied. The intercalators interfere with DNA replication or transcription resulted from intercalations of chomophores, thereby have the anticancer activity. According to previous studies, echinomycin is a bifunctional intercalating antitumor antibiotic which intercalates two quinoxaline rings into the bases of DNA duplex, and has a high binding affinity of DNA sequences rich in guanines and cytosines content. This study focuses on the DNA conformational changes and stability after binding of echinomycin to DNA with T:T mispair. We conducted a biophysical study to determine the effect of echinomycin binding to sequence having mispair. The stabilizing effects of Echi on DNA were characterized by UV meling. The result shows that echinomycin exhibits higher DNA-stablizing effect. The conformational changes were characterized using circular dichroism (CD) spectroscopy. We found the significant differences in the CD spectra after echinomycin bound to T:T mismatch sequence. It reveals that there is an obvious conformational effect of the complexes, and T:T mispair could form a more stable structure than echinomycin bound to other mispairs. Moreover, we solved the crystal structure of echinomycin bound to d(ACGTCG(BrdU))2. The current result shows that the DNA structure has conformational changes after binding of echinomycin to DNA. Because of the structural effects, it provides a favorable binding site for echinomycin. Afterwards we used surface plasmon resonance to analyze the kinetics, and calculate the binding affinities of echinomycin and different mismatched base pairs. Our result firmly establishes that echinomycin could elevate the stability of T:T mispair sequence. It also points out a useful direction for future new drugs design in the treatment of diseases related to T:T mispair.
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