A covalent protein-DNA 5'-product adduct is generated following AP lyase activity of human ALKBH1 (AlkB homologue 1).
A covalent protein-DNA 5'-product adduct is generated following AP lyase activity of human ALKBH1 (AlkB homologue 1).
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DOI：
10.1042/bj20121908
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发表时间：
2013-06-15
期刊：
影响因子：
--
通讯作者：
Hausinger RP
中科院分区：
文献类型：
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作者：
Müller TA;Andrzejak MM;Hausinger RP
ALKBH1 是哺乳动物 AlkB 同源物，具有脱碱基位点 (AP) 裂解酶活性。 AP 裂合酶反应通过活性位点 Lys 形成亚胺来催化，并且在 NaBH4 存在的情况下可以捕获共价中间体。令人惊讶的是，ALKBH1 在没有还原剂的情况下也能形成稳定的蛋白质-DNA 加合物。使用不同底物的实验表明该蛋白质与 5' DNA 产物共价结合；即含有α,β-不饱和醛的片段。 ALKBH1 的氨基末端结构域被确定为与 DNA 连接的主要位点。相比之下，诱变研究表明，形成亚胺键的主要催化残基是 Lys133，而该区域的 Lys154 和其他 Lys 残基则发挥机会作用。这些发现证实了 ALKBH1 作为 AP 裂合酶的分类，鉴定了参与裂合酶反应的初级和次级赖氨酸，并证明该蛋白质与 5' DNA 产物形成共价加合物。我们提出了两种可能的化学机制来解释共价连接。
ALKBH1 is a mammalian AlkB homolog that possesses abasic site (AP) lyase activity. The AP lyase reaction is catalyzed by imine formation with an active site Lys, and a covalent intermediate can be trapped in the presence of NaBH4. Surprisingly, ALKBH1 also forms a stable protein-DNA adduct in the absence of a reducing agent. Experiments with different substrates demonstrated that the protein covalently binds to the 5′ DNA product; i.e., the fragment containing an α,β-unsaturated aldehyde. The amino terminal domain of ALKBH1 was identified as the main site of linkage with DNA. By contrast, mutagenesis studies suggest that the primary catalytic residue forming the imine linkage is Lys133, with Lys154 and other Lys residues in this region serving in opportunistic roles. These findings confirm the classification of ALKBH1 as an AP lyase, identify the primary and a secondary Lys involved in the lyase reaction, and demonstrate the protein forms a covalent adduct with the 5′ DNA product. We propose two plausible chemical mechanisms to account for the covalent attachment.