Viscoelastic modeling of template-directed DNA synthesis.
In the present study, we have used the QCM-D technology to study the replication of surface attached oligonucleotide template strands using Escherichia coli DNA polymerase I (Klenow fragment, KF). Changes in resonance frequency (F) and energy dissipation (D) for DNA hybridization and polymerization were recorded at multiple harmonics. Formation of the polymerase/DNA complex led to a significant decrease in energy dissipation, which is consistent with a conformational change induced upon enzyme binding. This interpretation was further strengthened by a data analysis using a Voigt-based viscoelastic model. The analysis revealed a significant increase in shear viscosity and shear modulus during KF binding, whereas the viscoelastic properties of single- and double-stranded templates were almost identical. During the actual DNA synthesis, an initial increase in rigidity (shear viscosity) was followed by a gradual decrease that has two components corresponding to the release of enzyme and to the presence of the catalytically active enzyme/substrate complex. The corresponding decrease in surface concentration was found to underestimate the rate of enzyme release due to viscously coupled water that compensates for the loss in enzyme mass. Furthermore, the modeling elucidates that significant changes in both F and D originate from variations in the viscoelastic properties, which means that changes in F alone should be used with care for estimations of coupled mass and kinetics. Therefore, the modeled temporal variation in effective thickness, being proportional to coupled mass and, thus, independent of structural changes, was used to estimate the catalytic constants of the polymerization reaction. The reported work is the first example providing this type of structural information for the catalytic action of an enzyme, thereby demonstrating the potential of the technique for advanced analysis of complex biological reactions, including proper analysis of enzyme kinetics.
Stengel
Gudrun
G
Höök
Fredrik
F
Knoll
Wolfgang
W
D004269:DNA, Bacterial / Q000138:chemical synthesis* / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004566:Electrodes; D004926:Escherichia coli / Q000201:enzymology; D006046:Gold / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D009841:Oligonucleotides / Q000737:chemistry*; D011791:Quartz / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013499:Surface Properties; D013997:Time Factors; D014783:Viscosity
D004269:DNA, Bacterial; D009841:Oligonucleotides; D011791:Quartz; D006046:Gold; D012321:DNA-Directed RNA Polymerases