A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide election

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an impor-tant reaction of extraordinary complexity. The DNA polymerase does not merely facilitate the insertion of dNMP, but it also performs rapid screening of substrates to ensure a high degree of fidelity. Several kinetic studies have determined rate constants and equilibrium constants for the elementary steps that make up the overall pathway. The information is used to develop a macro-scopic kinetic model, using an approach described by Ninio [Ninio J., 1987. Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabili-ties. Proc. Natl. Acad. Sci. U.S.A. 84, 663–667]. The principle idea of the Ninio approach is to track a single template/primer complex over time and to identify the expected behavior. The average time to insert a single nucleotide is a weighted sum of several terms, in-cluding the actual time to insert a nucleotide plus delays due to polymerase detachment from ei-ther the ternary (template-primer-polymerase) or quaternary (+nucleotide) complexes and time delays associated with the identification and ultimate rejection of an incorrect nucleotide from the binding site. The passage times of all events and their probability of occurrence are ex-pressed in terms of the rate constants of the elementary steps of the reaction pathway. The model accounts for variations in the average insertion time with different nucleotides as well as the in-fluence of G+C content of the sequence in the vicinity of the insertion site. Furthermore the model provides estimates of error frequencies. If nucleotide extension is recognized as a compe-tition between successful insertions and time delaying events, it can be described as a binomial process with a probability distribution. The distribution gives the probability to extend a primer/template complex with a certain number of base pairs and in general it maps annealed complexes into extension products.

The Probe, Issue 229 – July/August 2003

• Human/Wildlife Conflicts in Ethiopia -- Jeff Peter sen, Berryman Institute, Utah State University • NADCA 2004 Election • If the Norway rat (Rattus norvegicus) originated in Asia, why is this rat commonly called a Norway rat? • Book Review: When Raccoons Fall through Your Ceiling: The Handbook for Coexisting with Wildlife by Andrea Dawn Lopez. • Antarctic Scientist Dies in Seal Attack: Research scientist, Kirsty Brown was snorkelling in waters close to the Rothera Research Station as part of her studies when a Leopard seal pulled her under. • Giant Lizards Spreading Through the Gulf Coast: Cape Coral, Florida has become a haven for Nile monitor lizards, and their population, which continues to grow, has possibly reached the thousands in the Gulf Coast city. • Monkeypox Outbreak: An Alert • NADCA 2004 Ballot