How a Low-Fidelity DNA Polymerase Chooses Non-Watson鈥揅rick from Watson鈥揅rick Incorporation

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• 作者：Wen-Jin Wu ; Mei-I Su ; Jian-Li Wu ; Sandeep Kumar ; Liang-hin Lim ; Chun-Wei Eric Wang ; Frank H. T. Nelissen ; Ming-Chuan Chad Chen ; Jurgen F. Doreleijers ; Sybren S. Wijmenga ; Ming-Daw Tsai
• 刊名：Journal of the American Chemical Society
• 出版年：2014
• 出版时间：April 2, 2014
• 年：2014
• 卷：136
• 期：13
• 页码：4927-4937
• 全文大小：668K
• 年卷期：v.136,no.13(April 2, 2014)
• ISSN：1520-5126

文摘

A dogma for DNA polymerase catalysis is that the enzyme binds DNA first, followed by MgdNTP. This mechanism contributes to the selection of correct dNTP by Watson鈥揅rick base pairing, but it cannot explain how low-fidelity DNA polymerases overcome Watson鈥揅rick base pairing to catalyze non-Watson鈥揅rick dNTP incorporation. DNA polymerase X from the deadly African swine fever virus (Pol X) is a half-sized repair polymerase that catalyzes efficient dG:dGTP incorporation in addition to correct repair. Here we report the use of solution structures of Pol X in the free, binary (Pol X:MgdGTP), and ternary (Pol X:DNA:MgdGTP with dG:dGTP non-Watson鈥揅rick pairing) forms, along with functional analyses, to show that Pol X uses multiple unprecedented strategies to achieve the mutagenic dG:dGTP incorporation. Unlike high fidelity polymerases, Pol X can prebind purine MgdNTP tightly and undergo a specific conformational change in the absence of DNA. The prebound MgdGTP assumes an unusual syn conformation stabilized by partial ring stacking with His115. Upon binding of a gapped DNA, also with a unique mechanism involving primarily helix 伪E, the prebound syn-dGTP forms a Hoogsteen base pair with the template anti-dG. Interestingly, while Pol X prebinds MgdCTP weakly, the correct dG:dCTP ternary complex is readily formed in the presence of DNA. H115A mutation disrupted MgdGTP binding and dG:dGTP ternary complex formation but not dG:dCTP ternary complex formation. The results demonstrate the first solution structural view of DNA polymerase catalysis, a unique DNA binding mode, and a novel mechanism for non-Watson鈥揅rick incorporation by a low-fidelity DNA polymerase.