The thermodynamics of template-directed DNA synthesis: Base insertion and extension enthalpies

Conceição A.S.A. Minetti, David P. Remeta, Holly Miller, Craig A. Gelfand, G. Eric Plum, Arthur P. Grollman, Kenneth J. Breslauer

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34 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14719-14724
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume100
Issue number25
DOIs
StatePublished - Dec 9 2003

All Science Journal Classification (ASJC) codes

  • General

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