Kaushik N, Pandey V N, Modak M J
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103, USA.
Biochemistry. 1996 Jun 4;35(22):7256-66. doi: 10.1021/bi960537i.
In order to identify functionally important residues in the O and O1 helices of Escherichia coli DNA polymerase I, we mutated 9 residues of this region to alanine. The alanine substitutions result in moderate to severe effects on the polymerase activity of the individual mutant enzymes. Severe loss of activity is associated with R754A, K758A, F762A, and Y766A. However, the loss of polymerase activity with different template primers exhibited a rather unique pattern implying differential participation of the individual residue in the synthesis directed by poly(rA), poly(dA), and poly(dC) templates. The ability of all mutants to form E-DNA binary complex was found to be unaffected with the exception of Y766A and F771A, where significant reduction in the cross-linking of both the template and the primer strand was noted. Most interestingly, the catalytic activity of all inactive mutant enzymes, with the exception of K758A, could be restored by substituting Mn2+ in place of Mg2+ as a divalent cation. Based on these results and associated changes in the kinetic parameters and other properties of the individual mutant enzyme, we conclude the following: (a) Tyr 766 and Phe 771 are either involved in the binding of template-primer or are in the vicinity of the DNA binding track. (b) Residues Arg 754, Lys 758, Phe 762, and Tyr 766 appear to be required for the binding of Mg.dTTP, while only Arg 754 and Lys 758 are utilized in the polymerization of Mn.dTTP. (c) In the polymerization of dGTP, only Lys 758 appears essential regardless of the type of divalent cation. (d) Phe 762 participates only in the binding of Mg.dTTP. Finally, (e) based on the analysis of the time course of nucleotide incorporation, processivity, and pyrophosphorolysis reaction, we suggest that Lys 758 is probably involved in a conformational change of the ternary complexes preceding and following the chemical step. In summary, our results suggest that the formation of the dNTP binding pocket is a dynamic process which requires the participation of different residues depending on the type of dNTP and the divalent cation.
为了鉴定大肠杆菌DNA聚合酶I的O螺旋和O1螺旋中功能重要的残基，我们将该区域的9个残基突变为丙氨酸。丙氨酸替代对各个突变酶的聚合酶活性产生中度至严重的影响。严重的活性丧失与R754A、K758A、F762A和Y766A相关。然而，不同模板引物导致的聚合酶活性丧失呈现出相当独特的模式，这意味着各个残基在由聚（rA）、聚（dA）和聚（dC）模板指导的合成中发挥不同的作用。除了Y766A和F771A，所有突变体形成E-DNA二元复合物的能力均未受影响，在Y766A和F771A中，模板链和引物链的交联显著减少。最有趣的是，除K758A外，所有无活性突变酶的催化活性都可以通过用Mn2+替代Mg2+作为二价阳离子来恢复。基于这些结果以及各个突变酶动力学参数和其他性质的相关变化，我们得出以下结论：（a）Tyr 766和Phe 771要么参与模板-引物的结合，要么位于DNA结合轨迹附近。（b）Arg 754、Lys 758、Phe 762和Tyr 766残基似乎是Mg.dTTP结合所必需的，而在Mn.dTTP聚合中仅利用了Arg 754和Lys 758。（c）在dGTP聚合中，无论二价阳离子的类型如何，似乎只有Lys 758是必需的。（d）Phe 762仅参与Mg.dTTP的结合。最后，（e）基于对核苷酸掺入时间进程、持续合成能力和焦磷酸解反应的分析，我们认为Lys 758可能参与了化学步骤前后三元复合物的构象变化。总之，我们的结果表明，dNTP结合口袋的形成是一个动态过程，根据dNTP的类型和二价阳离子的不同，需要不同残基的参与。
