Pelletier H, Sawaya M R, Kumar A, Wilson S H, Kraut J
Department of Chemistry, University of California, San Diego 92093-0317.
Science. 1994 Jun 24;264(5167):1891-903.
Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.
已分别在2.9埃和3.6埃分辨率下测定了大鼠DNA聚合酶β（pol β）、DNA模板引物和双脱氧胞苷三磷酸（ddCTP）的两种三元复合物。ddCTP是双脱氧胞苷（ddC）的三磷酸酯，ddC是一种核苷类似物，可靶向人类免疫缺陷病毒（HIV）的逆转录酶，目前用于治疗艾滋病。尽管这两种复合物的晶体属于不同的空间群，但结构相似，这表明聚合酶-DNA-ddCTP相互作用不受晶体堆积力的影响。在pol β活性位点，延伸引物的攻击3'-OH、ddCTP磷酸基团和两个Mg2+离子都聚集在天冬氨酸190、天冬氨酸192和天冬氨酸256周围。这些残基中的两个，天冬氨酸190和天冬氨酸256，存在于迄今为止研究的所有聚合酶的氨基酸序列中，并且在四种聚合酶（大肠杆菌DNA聚合酶I的Klenow片段、HIV-1逆转录酶、T7 RNA聚合酶和大鼠DNA pol β）中在空间上也相似，其晶体结构目前已知。描述了一种用于核苷酸转移反应的双金属离子机制，该机制可能适用于所有聚合酶。在所分析的三元复合物结构中，pol β与DNA模板引物的结合方式与最近提出的其他聚合酶-DNA模型不同。
