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The Thermodynamics of Template-Directed DNA Synthesis: Base Insertion and Extension Enthalpies

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The Thermodynamics of Template-Directed DNA Synthesis: Base Insertion and Extension Enthalpies The thermodynamics of template-directed DNA synthesis: Base insertion and extension enthalpies Conceic¸a˜ o A. S. A. Minetti*†, David P. Remeta*†, Holly Miller‡, Craig A. Gelfand*, G. Eric Plum*§, Arthur P. Grollman‡, and Kenneth J. Breslauer*¶ *Department of Chemistry and Chemical Biology, Rutgers, The State University of New Jersey, 610 Taylor Road, Piscataway, NJ 08854; and ‡Laboratory of Chemical Biology, Department of Pharmaceutical Sciences, Stony Brook University, Stony Brook, NY 11794 Communicated by I. M. Gelfand, Rutgers, The State University of New Jersey at New Brunswick, Piscataway, NJ, September 24, 2003 (received for review August 21, 2003) We used stopped-flow calorimetry to measure the overall enthalpy actions that preclude accurate determination of the relevant ther- change associated with template-directed nucleotide insertion and modynamic parameters. DNA extension. Specifically, we used families of hairpin self-priming Characterization of the energetics of DNA extension involves the templates in conjunction with an exonuclease-free DNA polymerase partitioning of a multitude of molecular events. These events to study primer extension by one or more dA or dT residues. Our include the initial polymerase–DNA association, formation of the results reveal exothermic heats between ؊9.8 and ؊16.0 kcal͞bp for productive ternary complex with the incoming nucleotide, and the template-directed enzymatic polymerization. These extension enthal- chemical processes culminating in extension of the oligonucleotide pies depend on the identity of the inserting base, the primer terminus, chain. In this multistep recognition and association process (3, 18), and͞or the preceding base. Despite the complexity of the overall exoϪ it has been proposed that KF , in complex with the template– process, the sign, magnitude, and sequence dependence of these primer duplex, undergoes a conformational change on interaction insertion and extension enthalpies are consistent with nearest- with the incoming nucleotide, resulting in formation of the active neighbor data derived from DNA melting studies. We recognize that closed ternary complex (2, 5, 17). After nucleotidyl transfer and the overall process studied here involves contributions from a mul- phosphodiester bond formation, a second conformational change titude of events, including dNTP to dNMP hydrolysis, phosphodiester that results in relaxation of the complex and release of pyrophos- bond formation, and enzyme conformational changes. It is therefore phate has been reported (5). Structural insight into the KFexoϪ noteworthy that the overall enthalpic driving force per base pair is of ternary complex has been reported (17), and the overall process has a magnitude similar to that expected for addition of one base pair or been reviewed in terms of transition-state mechanisms (19). base stack per insertion event, rather than that associated with the Numerous studies of polymerase-catalyzed template-directed rupture and͞or formation of covalent bonds, as occurs during this DNA synthesis have evaluated the kinetic parameters for canonical catalytic process. Our data suggest a constant sequence-independent background of compensating enthalpic contributions to the overall extension, the preferences for misinsertions, and the characteristics process of DNA synthesis, with discrimination expressed by differ- of translesion synthesis. In the aggregate, these studies reveal ences in noncovalent interactions at the template–primer level. Such canonical and noncanonical extension to be strongly dependent enthalpic discrimination underscores a model in which complex bio- on the subtleties of the neighboring sequences comprising the logical events are regulated by relatively modest energy balances template–primer region of the target DNA, as well as on the nature involving weak interactions, thereby allowing subtle mechanisms of of the polymerase and the lesion (20–28). Consequently, the regulation. surrounding sequence context plays a critical role in the mutage- nicity of a particular DNA lesion (26, 29, 30). Energetic character- ization of the sequence-dependent extension events is needed to wealth of structural and biochemical information has provided understand the nature and magnitude of the forces that dictate and significant insight into the mechanisms of DNA polymerase BIOCHEMISTRY A control template-directed nucleic acid synthesis. activity (1–8). Kinetic and structural studies on a number of In recognition of this need, the energetics of nucleic acid polymerase systems (9–18) have furnished valuable information regarding the molecular mechanisms governing DNA replication. synthesis has been the focus of intense interest, nearly concomitant Despite the considerable efforts that have been undertaken to with the discovery of the DNA helical structure. Among the characterize the overall catalytic process, there is a paucity of pioneers in the field, Peller and coworkers (e.g., ref. 31) dedicated thermodynamic data describing the energetic parameters associ- significant efforts toward elucidating the thermodynamic basis for ated with DNA chain elongation. Such studies are essential to nucleic acid synthesis by employing noncalorimetric techniques. A elucidate the nature and magnitude of the forces that control the number of other studies have invoked thermodynamic models mechanisms of polymerase-mediated recognition and catalysis based on biochemical and kinetic approaches (32–34) to elucidate associated with template-directed nucleic acid synthesis. the origins of the high fidelity observed in DNA synthesis. The use Escherichia coli DNA polymerase I has been explored extensively of calorimetric techniques to assess enzyme kinetic and energetic in terms of its structural (9) and biochemical (3) properties. The parameters has long been proposed by Sturtevant and has been Klenow fragment (KF) is a 68-kDa polypeptide catalytic domain of practiced since the early 1960s (35, 36). Only recently, however, this enzyme that retains full polymerase and 3Ј–5Ј exonuclease have advances in calorimetric instrumentation (with sufficient activity. To eliminate proofreading and editing that results in sensitivity and resolution) and molecular biology and chemistry degradation of the DNA product, it is desirable to employ an techniques allowed quantitative studies on the energetics of bio- exonuclease-deficient KF (KFexoϪ) in biochemical and biophysical molecular synthesis and recognition events. studies. Such a system has been obtained by selective site-directed mutations (D355A and E357A), which effectively abrogate the exoϪ 3Ј–5Ј exonuclease activities (12) while retaining full polymerase Abbreviations: KF, Klenow fragment; KF , exonuclease-deficient KF. activity (9). The availability of the KFexoϪ provides an ideal system †C.A.S.A.M. and D.P.R. contributed equally to this work. to explore polymerase-catalyzed insertion and extension energetics §Present address: IBET, Inc., Upper Arlington, OH 43220. because it permits characterization of both correct insertions and ¶To whom correspondence should be addressed. E-mail: [email protected]. potential misincorporations while being devoid of concurrent re- © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2336142100 PNAS ͉ December 9, 2003 ͉ vol. 100 ͉ no. 25 ͉ 14719–14724 Downloaded by guest on September 24, 2021 In this article, we report use of an ultrasensitive differential removed by extensive lyophilization involving multiple resuspen- stopped-flow calorimeter to measure reaction heats associated with sions in water. This purification͞lyophilization protocol effectively polymerase-mediated template-directed correct base insertion and inhibits disproportionation of the nucleotides before calorimetric extension. In addition to their intrinsic value, these data also measurement. provide a baseline for the dissection of the energetic landscape associated with both normal synthesis and patterns of mutagenesis Temperature-Dependent UV Spectroscopy. An aliquot of each oligo- and translesion synthesis. nucleotide stock solution was diluted in the standard reagent buffer, composed of 50 mM Pipes and 5.0 mM MgCl2 (pH 7.4), to a Materials and Methods ␮ Ϫ concentration of 10 M. Thermal melting profiles were monitored DNA Polymerase. The KFexo of E. coli DNA polymerase I (37) has at 260 nm in an AVIV 14DS UV͞Vis spectrophotometer (Aviv been overexpressed and purified as described (9). The lack of Biomedical, Lakewood, NJ) over the temperature range of 0–100°C detectable exonuclease activity (over incubation periods as long as by employing a 10-sec integration period at 0.5°C increments. Ϫ 48 h) has been ensured by gel electrophoresis analysis of KFexo Repetitive optical profiles revealed monophasic concentration- preparations with DNA. The integrity of the enzyme preparations independent melting behavior, consistent with the thermally in- in terms of both activity and stability was verified under the solution duced disruption of hairpin structures. conditions selected for calorimetric measurement of template- directed DNA synthesis. Differential Stopped-Flow Calorimetry. Heats associated with tem- plate-directed DNA synthesis were measured in a differential Deoxyoligonucleotide Templates. Oligonucleotides designed to form stopped-flow heat conduction calorimeter (Commonwealth Tech- hairpin structures containing an overhang of one to five nucleotide nology, Alexandria, VA), which is described in detail elsewhere repeats (Table
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