Saturno J, Lázaro J M, Esteban F J, Blanco L, Salas M
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain.
J Mol Biol. 1997 Jun 13;269(3):313-25. doi: 10.1006/jmbi.1997.1053.
Bacteriophage ø29 DNA polymerase shares with other DNA-dependent DNA polymerases several regions of amino acid homology along the primary structure. Among them, motif B, characterized by the consensus +x3Kx(6-7)YG (+ being a positively charged amino acid), appears to be specifically conserved in those polymerases that use DNA but not RNA as template. In particular, the lysine residue of this motif is invariant in all members of DNA-dependent polymerases. In this paper we report a mutational analysis of this invariant residue of motif B with the construction and characterization of two mutant proteins in the corresponding residue (Lys383) of ø29 DNA polymerase. Mutant proteins (K383R and K383P) were overexpressed, purified and analyzed under steady-state conditions. In agreement with the modular organization proposed for ø29 DNA polymerase, the exonuclease activity was not affected in either mutant protein. Conversely, mutant K383P showed no detectable capacity to incorporate dNTP substrates using either DNA or TP as primer, although its affinity for DNA was not affected. The conservative substitution of Lys383 by arginine (K383R) resulted in a considerable impairment to use dNTPs, in both processive and non-processive DNA synthesis; the Km for dNTPs being 200-fold higher than that of the wild-type enzyme. Mutant K383R recovered the wild-type polymerase/exonuclease ratio when Mn2+ was used instead of Mg2+ as metal activator, indicating a distorted binding of the [dNTP-metal] chelate at the mutant enzyme active site. The positive charge at residue Lys383 was also critical in the catalysis of deoxynucleotidylation of the terminal protein by ø29 DNA polymerase. The results obtained suggest a direct role for the lysine residue in motif B in forming an evolutionarily conserved DNA templated dNTP binding pocket. Additionally, K383R mutant protein was also affected in the progression from protein-primed initiation to DNA elongation, a switch between two modes of priming that characterizes ø29 DNA replication.
噬菌体φ29 DNA聚合酶在一级结构上与其他依赖DNA的DNA聚合酶有几个氨基酸同源区域。其中，基序B的特征是共有序列+x3Kx(6 - 7)YG（+为带正电荷的氨基酸），似乎在那些以DNA而非RNA为模板的聚合酶中特别保守。特别是，该基序中的赖氨酸残基在所有依赖DNA的聚合酶成员中都是不变的。在本文中，我们报告了对基序B中这个不变残基的突变分析，构建并表征了φ29 DNA聚合酶相应残基（Lys383）处的两种突变蛋白。突变蛋白（K383R和K383P）被过量表达、纯化并在稳态条件下进行分析。与为φ29 DNA聚合酶提出的模块化组织一致，两种突变蛋白的核酸外切酶活性均未受影响。相反，突变体K383P无论是使用DNA还是TP作为引物，都没有检测到掺入dNTP底物的能力，尽管其对DNA的亲和力未受影响。用精氨酸保守取代Lys383（K383R）导致在进行性和非进行性DNA合成中使用dNTP的能力大幅受损；dNTP的Km比野生型酶高200倍。当使用Mn2+而非Mg2+作为金属激活剂时，突变体K383R恢复了野生型聚合酶/核酸外切酶比率，表明在突变酶活性位点处[dNTP - 金属]螯合物的结合发生了扭曲。残基Lys383处的正电荷在φ29 DNA聚合酶催化末端蛋白的脱氧核苷酸化中也很关键。获得的结果表明基序B中的赖氨酸残基在形成进化保守的DNA模板dNTP结合口袋中起直接作用。此外，K383R突变蛋白在从蛋白质引发起始到DNA延伸的过程中也受到影响，这是φ29 DNA复制的两种引发模式之间的转换。
