Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed β-elimination at abasic sites
Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed β-elimination at abasic sites
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DOI：
10.1021/bi020026y
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发表时间：
2002-06-04
期刊：
影响因子：
2.9
通讯作者：
Lloyd, RS
中科院分区：
文献类型：
--
作者：
Kurtz, AJ;Dodson, ML;Lloyd, RS
先前的研究表明，含有单个芳香残基的肽，如Lys-Trp-Lys，可以通过β消除在AP位点切割双工DNA的磷酸二酯主链。胺作为反应性亲核试剂攻击开环脱氧核糖上的cv，形成瞬时肽- dna亚胺（希夫碱）中间体，在还原条件下可作为稳定的共价物质分离。在目前的研究中，我们使用这种方法来证明肽催化的β消除是通过两个希夫碱中间体的形成进行的，其中一个在链切割之前被共价捕获，另一个在链切割之后被捕获。活性肽n端乙酰化显著抑制捕获希夫碱络合物的形成；因此，我们首次证明了肽催化链切割的首选反应性亲核试剂是n端ε -氨基，而不是先前假设的位于赖氨酸残基上的ε -氨基。在中心色氨酸残基转变为丙氨酸的捕获反应中，对希夫碱形成的效率没有显著影响，这表明芳香残基的存在对于肽催化的β消除之前的步骤是必不可少的。有趣的是，本文提出的方法提供了一种方便的方法，以特定位点的方式将一组肽共价连接到含有AP位点的DNA上。我们认为，这种dna -肽交联的产生可能为研究具有生物学意义的dna -蛋白交联损伤的修复提供实用价值，因为dna -肽复合物可能模仿dna -蛋白交联修复途径中的中间结构。
Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via beta-elimination. An amine serves as the reactive nucleophile to attack C V on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed beta-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal epsilon-amino group, not an epsilon-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to atanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed beta-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.