Spratt T E
American Health Foundation, 1 Dana Road, Valhalla, New York 10595, USA.
Biochemistry. 2001 Mar 6;40(9):2647-52. doi: 10.1021/bi002641c.
DNA polymerases replicate DNA with high fidelity despite the small differences in energy between correct and incorrect base pairs. X-ray crystallographic and structure-activity kinetic experiments have implicated interactions with the minor groove of the DNA as being crucial for catalysis and fidelity. The current hypothesis is that polymerases check the geometry of the base pairs through hydrogen bonds and steric interactions with the minor groove of the DNA. The mechanisms by which these interactions are related to catalysis and fidelity are not known. In this manuscript, we have studied these interactions using a combination of site-specific mutagenesis of Escherichia coli DNA polymerase I (Klenow fragment) and atomic substitution of the DNA. Crystal structures have predicted hydrogen bonds from Arg668 to the terminal base on the primer (P1) and Gln849 to its base pair partner (T1). Kinetic studies, however, have implicated the minor groove of the primer terminus but not its base pair partner as being important to catalysis and fidelity. Hydrogen bonds between Arg668 and Gln849 to the DNA were probed with the site specific mutants, R668A and Q849A. Hydrogen bonds from the DNA were probed with three oligodeoxynucleotides which have a guanine or 3-deazaguanine (3DG) at P1, T1, or T2. We found that the pre-steady-state parameter k(pol) was decreased with R668A (40-fold) and Q849A (150-fold) or with 3DG at P1 (300-fold) or T2 (25-fold). When R668A was combined with 3DG at P1 the decrease in rate was only 80-fold, consistent with a hydrogen bond between Arg668 and P1. In contrast, when the 3DG substitution at P1 was combined with Q849A the rate reduction was 15000-fold. Similar reactions between R668A or Q849A and T2 showed that there are interactions between these sites although the interactions are not as strong as between P1 and R668.
尽管正确和错误碱基对之间的能量差异很小，DNA聚合酶仍能以高保真度复制DNA。X射线晶体学和结构-活性动力学实验表明，与DNA小沟的相互作用对催化和保真度至关重要。目前的假设是，聚合酶通过与DNA小沟的氢键和空间相互作用来检查碱基对的几何形状。这些相互作用与催化和保真度相关的机制尚不清楚。在本论文中，我们结合大肠杆菌DNA聚合酶I（克列诺片段）的位点特异性诱变和DNA的原子取代来研究这些相互作用。晶体结构预测了从Arg668到引物上的末端碱基（P1）以及从Gln849到其碱基对配对体（T1）的氢键。然而，动力学研究表明，引物末端的小沟而非其碱基对配对体对催化和保真度很重要。用位点特异性突变体R668A和Q849A探测了Arg668和Gln849与DNA之间的氢键。用三种在P1、T1或T2处含有鸟嘌呤或3-脱氮鸟嘌呤（3DG）的寡脱氧核苷酸探测了来自DNA的氢键。我们发现，R668A（40倍）和Q849A（150倍）或P1处的3DG（300倍）或T2处的3DG（25倍）会使稳态前参数k(pol)降低。当R668A与P1处的3DG结合时，速率降低仅80倍，这与Arg668和P1之间的氢键一致。相反，当P1处的3DG取代与Q849A结合时，速率降低为15000倍。R668A或Q849A与T2之间的类似反应表明，这些位点之间存在相互作用，但这种相互作用不如P1和R668之间的强。
