Electronic Measurements of Single-Molecule Processing by DNA Polymerase I (Klenow Fragment)

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• 作者：Tivoli J. Olsen ; Yongki Choi ; Patrick C. Sims ; O. Tolga Gul ; Brad L. Corso ; Chengjun Dong ; William A. Brown ; Philip G. Collins ; Gregory A. Weiss
• 刊名：The Journal of the American Chemical Society
• 出版年：2013
• 出版时间：May 29, 2013
• 年：2013
• 卷：135
• 期：21
• 页码：7855-7860
• 全文大小：331K
• 年卷期：v.135,no.21(May 29, 2013)
• ISSN：1520-5126

文摘

Bioconjugating single molecules of the Klenow fragment of DNA polymerase I into electronic nanocircuits allowed electrical recordings of enzymatic function and dynamic variability with the resolution of individual nucleotide incorporation events. Continuous recordings of DNA polymerase processing multiple homopolymeric DNA templates extended over 600 s and through >10鈥?00 bond-forming events. An enzymatic processivity of 42 nucleotides for a template of the same length was directly observed. Statistical analysis determined key kinetic parameters for the enzyme鈥檚 open and closed conformations. Consistent with these nanocircuit-based observations, the enzyme鈥檚 closed complex forms a phosphodiester bond in a highly efficient process >99.8% of the time, with a mean duration of only 0.3 ms for all four dNTPs. The rate-limiting step for catalysis occurs during the enzyme鈥檚 open state, but with a nearly 2-fold longer duration for dATP or dTTP incorporation than for dCTP or dGTP into complementary, homopolymeric DNA templates. Taken together, the results provide a wealth of new information complementing prior work on the mechanism and dynamics of DNA polymerase I.