Mizrahi V, Huberts P
Molecular Biology Unit, South African Institute for Medical Research and Department of Hematology, University of the Witwatersrand Medical School, Johannesburg, South Africa.
Nucleic Acids Res. 1996 Dec 15;24(24):4845-52. doi: 10.1093/nar/24.24.4845.
The DNA polymerase I (PolI) from Mycobacterium tuberculosis (Mtb) was overproduced in Escherichia coli as an enzymatically active, recombinant protein with or without an N-terminal His-tag. The proteins catalysed both the DNA polymerisation of homo- and heteropolymer template-primers and the 5'-3' exonucleolytic hydrolysis of gapped and nicked substrates but lacked an associated proofreading activity. In accordance with recent predictions [Tabor, S. and Richardson, C.C. (1995) Proc. Natl. Acad. Sci. USA, 92, 6339-6343], both recombinant forms of the M. tuberculosis enzyme were unable to discriminate against dideoxynucleotide 5'-triphosphates and were thus efficiently inhibited by these chain-terminating nucleotide analogues during DNA synthesis. This unusual property might be potentially exploitable in terms of novel anti-mycobacterial drug design. A mutational analysis of 5' nuclease domain residues allowed the roles of nine invariant acidic residues to be evaluated. Acidic side chain neutralisation resulted in a > or = 20-fold reduction in activity, with the most profound reduction (> or = 10(4)-fold) being caused by neutralisation of the Asp125, Asp148 and Asp150 residues.
结核分枝杆菌（Mtb）的DNA聚合酶I（PolI）在大肠杆菌中过量表达，产生了具有或不具有N端His标签的、具有酶活性的重组蛋白。这些蛋白既能催化同聚物和杂聚物模板引物的DNA聚合反应，也能催化缺口和切口底物的5'-3'核酸外切酶水解反应，但缺乏相关的校对活性。根据最近的预测[Tabor, S.和Richardson, C.C.（1995年）《美国国家科学院院刊》，92, 6339-6343]，结核分枝杆菌酶的两种重组形式都无法区分双脱氧核苷酸5'-三磷酸，因此在DNA合成过程中被这些链终止核苷酸类似物有效抑制。就新型抗分枝杆菌药物设计而言，这种不寻常的特性可能具有潜在的利用价值。对5'核酸酶结构域残基的突变分析使得九个不变酸性残基的作用得以评估。酸性侧链中和导致活性降低≥20倍，其中Asp125、Asp148和Asp150残基的中和导致活性降低最为显著（≥10^4倍）。
