Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase
Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase
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DOI：
10.1093/nar/30.5.1198
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发表时间：
2002-03-01
影响因子：
14.9
通讯作者：
Pommier, Y
中科院分区：
文献类型：
--
作者：
Debéthune, L;Kohlhagen, G;Pommier, Y
酪氨酸二酯酶-1(Tdp1)是目前已知的唯一一种从DNA片段3‘端连接氨基酸的复合体中去除酪氨酸的酶。这种复合体可以在拓扑异构酶I处理DNA后产生，最近在酵母中的研究证明了Tdp1在拓扑异构酶I介导的DNA损伤后对细胞生存的重要性。在本研究中，我们使用人工合成的寡核苷酸-多肽结合物(核肽)和重组酵母Tdp1来研究Tdp1活性的分子决定因素。我们发现Tdp1可以处理多达13个氨基酸残基的核肽，但与70 kDa的拓扑异构酶I共价连接到自杀DNA底物上时活性较差。此外，Tdp1对含有1-4个氨基酸的核多肽比15个氨基酸的核多肽更有效。含15个核苷酸的核多肽对Tdp1的作用也比含4个核苷酸的同源核多肽更有效。这些结果表明，DNA结合有助于Tdp1的活性，并且Tdp1在体内拓扑异构酶I被蛋白降解后最有效。
Tyrosyl-DNA phosphodiesterase-1 (Tdp1) is the only known enzyme to remove tyrosine from complexes in which the amino acid is linked to the 3'-end of DNA fragments. Such complexes can be produced following DNA processing by topoisomerase I, and recent studies in yeast have demonstrated the importance of TDP1 for cell survival following topoisomerase I-mediated DNA damage. In the present study, we used synthetic oligodeoxynucleotide-peptide conjugates (nucleopeptides) and recombinant yeast Tdp1 to investigate the molecular determinants for Tdp1 activity. We find that Tdp1 can process nucleopeptides with up to 13 amino acid residues but is poorly active with a 70 kDa fragment of topoisomerase I covalently linked to a suicide DNA substrate. Furthermore, Tdp1 was more effective with nucleopeptides with one to four amino acids than 15 amino acids. Tdp1 was also more effective with nucleopeptides containing 15 nt than with homolog nucleopeptides containing 4 nt. These results suggest that DNA binding contributes to the activity of Tdp1 and that Tdp1 would be most effective after topoisomerase I has been proteolyzed in vivo.