Catalysis of DNA cleavage and nucleoside triphosphate synthesis by NM23-H2/NDP kinase share an active site that implies a DNA repair function
Catalysis of DNA cleavage and nucleoside triphosphate synthesis by NM23-H2/NDP kinase share an active site that implies a DNA repair function
复制标题
DOI：
10.1073/pnas.97.26.14194
复制
发表时间：
2000-12-19
影响因子：
11.1
通讯作者：
Kyin, S
中科院分区：
文献类型：
--
作者：
Postel, EH;Abramczyk, BM;Kyin, S
NM23/NDP激酶在发育和癌症中发挥重要作用，但其生物学功能尚不清楚，尽管其生物化学特性包括核苷二磷酸激酶（NDP激酶），DNA结合和转录，突变子功能以及通过共价酶-DNA复合物切割异常结构的DNA。为了评估核酸酶在人类NM23-H2中的作用，我们试图确定负责共价催化的氨基酸。通过测序DNA连接肽和定点诱变。我们确定了赖氨酸-12，一个系统发育上保守的残基，作为与DNA形成共价复合物的氨基酸。特别是，e -氨基作为关键的亲核试剂，因为与谷氨酰胺取代而不是精氨酸完全废除了共价加合物的形成和DNA切割，而DNA结合特性保持不变。这些发现和化学修饰数据表明，磷酸二酯键的切割是通过一种DNA糖基酶/裂解酶样机制发生的，这种机制被称为碱基切除DNA修复核酸酶的特征。NM23/NDP激酶参与DNA修复途径与其在正常和肿瘤细胞发育中的作用一致。此外，在x射线晶体结构中已知位于NDP激酶磷酸化反应的催化包中的赖氨酸-12，也被发现对NM23-H2的NDP激酶活性至关重要，这表明NM23-H2的两种催化活性具有根本联系。
NM23/NDP kinases play an important role in development and cancer but their biological function is unknown, despite an intriguing collection of biochemical properties including nucleoside-diphosphate kinase (NDP kinase), DNA binding and transcription, a mutator function, and cleavage of unusually structured DNA by means of a covalent enzyme-DNA complex. To assess the role of the nuclease in human NM23-H2, we sought to identify the amino acid responsible for covalent catalysis. By sequencing a DNA linked peptide and by site-directed mutagenesis. we identified lysine-12, a phylogenetically conserved residue, as the amino acid forming the covalent complex with DNA. In particular, the E-amino group acts as the critical nucleophile, because substitution with glutamine but not arginine completely abrogated covalent adduct formation and DNA cleavage, whereas the DNA-binding properties remained intact. These findings and chemical modification data suggest that phosphodiester-bond cleavage occurs by a DNA glycosylase/lyase-like mechanism known as the signature of base excision DNA repair nucleases. Involvement of NM23/NDP kinase in a DNA repair pathway would be consistent with its role in normal and tumor cell development. Additionally, lysine-12, which is known in the x-ray crystallographic structure to lie in the catalytic packet involved in the NDP kinase phosphorylation reaction, was found essential also for the NDP kinase activity of NM23-H2, suggesting that the two catalytic activities of NM23-H2 are fundamentally connected.