文章基本信息

标题：The thermodynamics of template-directed DNA synthesis: Base insertion and extension enthalpies
本地全文：下载
作者：Conceição A. S. A. Minetti ; David P. Remeta ; Holly Miller 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2003
卷号：100
期号：25
页码：14719-14724
DOI：10.1073/pnas.2336142100
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We used stopped-flow calorimetry to measure the overall enthalpy change associated with template-directed nucleotide insertion and DNA extension. Specifically, we used families of hairpin self-priming templates in conjunction with an exonuclease-free DNA polymerase to study primer extension by one or more dA or dT residues. Our results reveal exothermic heats between -9.8 and -16.0 kcal/bp for template-directed enzymatic polymerization. These extension enthalpies depend on the identity of the inserting base, the primer terminus, and/or the preceding base. Despite the complexity of the overall process, the sign, magnitude, and sequence dependence of these insertion and extension enthalpies are consistent with nearest-neighbor data derived from DNA melting studies. We recognize that the overall process studied here involves contributions from a multitude of events, including dNTP to dNMP hydrolysis, phosphodiester bond formation, and enzyme conformational changes. It is therefore noteworthy that the overall enthalpic driving force per base pair is of a magnitude similar to that expected for addition of one base pair or base stack per insertion event, rather than that associated with the rupture and/or formation of covalent bonds, as occurs during this catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall process of DNA synthesis, with discrimination expressed by differences in noncovalent interactions at the template-primer level. Such enthalpic discrimination underscores a model in which complex biological events are regulated by relatively modest energy balances involving weak interactions, thereby allowing subtle mechanisms of regulation.