### Abstract

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an important 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 macroscopic 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 probabilities. 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, including the actual time to insert a nucleotide plus delays due to polymerase detachment from either 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 expressed 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 influence 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 competition 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.

Original language | English (US) |
---|---|

Pages (from-to) | 101-110 |

Number of pages | 10 |

Journal | Computational Biology and Chemistry |

Volume | 29 |

Issue number | 2 |

DOIs | |

State | Published - Apr 1 2005 |

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### Keywords

- Biochemical engineering
- Mathematical model
- Molecular biology
- Polymerase chain reaction

### ASJC Scopus subject areas

- Biochemistry
- Structural Biology
- Analytical Chemistry
- Physical and Theoretical Chemistry

### Cite this

**A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection.** / Viljoen, Steve; Griep, Mark A; Nelson, Michael; Viljoen, Hendrik J.

Research output: Contribution to journal › Article

*Computational Biology and Chemistry*, vol. 29, no. 2, pp. 101-110. https://doi.org/10.1016/j.compbiolchem.2005.02.003

}

TY - JOUR

T1 - A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection

AU - Viljoen, Steve

AU - Griep, Mark A

AU - Nelson, Michael

AU - Viljoen, Hendrik J

PY - 2005/4/1

Y1 - 2005/4/1

N2 - The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an important 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 macroscopic 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 probabilities. 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, including the actual time to insert a nucleotide plus delays due to polymerase detachment from either 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 expressed 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 influence 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 competition 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.

AB - The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an important 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 macroscopic 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 probabilities. 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, including the actual time to insert a nucleotide plus delays due to polymerase detachment from either 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 expressed 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 influence 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 competition 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.

KW - Biochemical engineering

KW - Mathematical model

KW - Molecular biology

KW - Polymerase chain reaction

UR - http://www.scopus.com/inward/record.url?scp=16344368485&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=16344368485&partnerID=8YFLogxK

U2 - 10.1016/j.compbiolchem.2005.02.003

DO - 10.1016/j.compbiolchem.2005.02.003

M3 - Article

VL - 29

SP - 101

EP - 110

JO - Computational Biology and Chemistry

JF - Computational Biology and Chemistry

SN - 1476-9271

IS - 2

ER -