The thermodynamics of template-directed DNA synthesis: base insertion and extension enthalpies.
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.
Minetti
Conceição A S A
CA
Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854, USA.
Remeta
David P
DP
Miller
Holly
H
Gelfand
Craig A
CA
Plum
G Eric
GE
Grollman
Arthur P
AP
Breslauer
Kenneth J
KJ
D001483:Base Sequence; D002151:Calorimetry; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D019277:Entropy; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D013696:Temperature; D013698:Templates, Genetic; D013816:Thermodynamics; D014466:Ultraviolet Rays
D009841:Oligonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase