Singh Kamalendra, Modak Mukund J
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
Biochemistry. 2005 Jun 7;44(22):8101-10. doi: 10.1021/bi050140r.
Previous structural and biochemical data indicate a participation of the J-helix of Escherichia coli pol I in primer positioning at the polymerase and exonuclease sites. The J-helix contains three polar residues: N675, Q677, and N678. Preliminary characterization of alanine substitutions of these residues showed that only Q677A DNA polymerase has substantially decreased polymerase and increased exonuclease activity. The Q677A enzyme had approximately 2- and approximately 5-fold greater exonuclease activity than the wild type (WT) with mismatched and matched template-primers (TPs), respectively. N675A and N678A DNA polymerases did not differ significantly from the WT in these activities, despite the fact that both residues are seen to interact with the TP in various pol I-DNA complexes. Pre-steady-state kinetic measurements for the exonuclease activity of WT and mutant enzymes indicated nearly identical DNA binding affinity for ssDNA and mismatched TPs. However, with a matched TP, Q677A DNA polymerase exhibited increased exonuclease site affinity. The most important characteristic of Q677A DNA polymerase was its ability to continue cleavage into the matched region of the TP after mismatch excision, in contrast to the WT and other mutant enzymes. The increase in the exonuclease activity of Q677A DNA polymerase was further determined not to be solely due to the weakened binding at the polymerase site, by comparison with another polymerase-defective mutant enzyme, namely, R668A DNA polymerase. These enzymes have significantly decreased DNA binding affinity at the polymerase site, yet the exonuclease activity parameters of R668A DNA polymerase remain similar to those of the WT. These results strongly suggest that participation of Q677 is required for positioning the primer terminus (a) in the polymerase site for continued nucleotide addition and (b) in the 3'-exonuclease site for the controlled removal of mismatched nucleotides.
先前的结构和生化数据表明，大肠杆菌DNA聚合酶I的J螺旋参与了引物在聚合酶和核酸外切酶位点的定位。J螺旋包含三个极性残基：N675、Q677和N678。对这些残基的丙氨酸替代进行的初步表征表明，只有Q677A DNA聚合酶的聚合酶活性大幅降低，核酸外切酶活性增加。与野生型（WT）相比，Q677A酶在错配和匹配模板引物（TP）时的核酸外切酶活性分别高出约2倍和约5倍。尽管在各种DNA聚合酶I-DNA复合物中都观察到N675和N678这两个残基与TP相互作用，但N675A和N678A DNA聚合酶在这些活性方面与WT没有显著差异。对WT和突变酶的核酸外切酶活性进行的稳态前动力学测量表明，它们对单链DNA和错配TP的DNA结合亲和力几乎相同。然而，对于匹配的TP，Q677A DNA聚合酶表现出更高的核酸外切酶位点亲和力。与WT和其他突变酶相比，Q677A DNA聚合酶最重要的特征是其在错配切除后能够继续切割进入TP的匹配区域。通过与另一种聚合酶缺陷型突变酶R668A DNA聚合酶比较，进一步确定Q677A DNA聚合酶核酸外切酶活性的增加并非仅仅由于其在聚合酶位点的结合减弱。这些酶在聚合酶位点的DNA结合亲和力显著降低，但R668A DNA聚合酶的核酸外切酶活性参数仍与WT相似。这些结果强烈表明，Q677的参与对于将引物末端定位在（a）聚合酶位点以继续添加核苷酸和（b）3'-核酸外切酶位点以可控地去除错配核苷酸是必需的。
