Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.

Published

Journal Article

We used a series of dATP and dGTP analogues to determine how DNA polymerase I from Bacillus stearothermophilus (BF), a prototypical A family polymerase, uses N-1, N(2), N-3, and N(6) of purine dNTPs to differentiate between right and wrong nucleotide incorporation. Altering any of these nitrogens had two effects. First, it decreased the efficiency of correct incorporation of the resulting dNTP analogue, with the loss of N-1 and N-3 having the most severe effects. Second, it dramatically increased the rate of misincorporation of the resulting dNTP analogues, with alterations in either N-1 or N(6) having the most severe impacts. Adding N(2) to dNTPs containing the bases adenine and purine increased the degree of polymerization opposite T but also tremendously increased the degree of misincorporation opposite A, C, and G. Thus, BF uses N-1, N(2), N-3, and N(6) of purine dNTPs both as negative selectors to prevent misincorporation and as positive selectors to enhance correct incorporation. Comparing how BF discriminates between right and wrong dNTPs with both B family polymerases and low-fidelity polymerases indicates that BF has chosen a unique solution vis-a-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.

Full Text

Duke Authors

Cited Authors

  • Trostler, M; Delier, A; Beckman, J; Urban, M; Patro, JN; Spratt, TE; Beese, LS; Kuchta, RD

Published Date

  • June 2, 2009

Published In

Volume / Issue

  • 48 / 21

Start / End Page

  • 4633 - 4641

PubMed ID

  • 19348507

Pubmed Central ID

  • 19348507

Electronic International Standard Serial Number (EISSN)

  • 1520-4995

Digital Object Identifier (DOI)

  • 10.1021/bi900104n

Language

  • eng

Conference Location

  • United States