Abstract
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, N2, N-3, and N6 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 N6 having the most severe impacts. Adding N2 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, N2, N-3, and N6 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-à-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4633-4641 |
| Number of pages | 9 |
| Journal | Biochemistry |
| Volume | 48 |
| Issue number | 21 |
| DOIs | |
| State | Published - Jun 2 2009 |
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All Science Journal Classification (ASJC) codes
- Biochemistry
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Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus. / Trostler, Michael; Delier, Alison; Beckman, Jeff; Urban, Milan; Patro, Jennifer N.; Spratt, Thomas; Beese, Lorena S.; Kuchta, Robert D.
In: Biochemistry, Vol. 48, No. 21, 02.06.2009, p. 4633-4641.Research output: Contribution to journal › Article
TY - JOUR
T1 - Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus
AU - Trostler, Michael
AU - Delier, Alison
AU - Beckman, Jeff
AU - Urban, Milan
AU - Patro, Jennifer N.
AU - Spratt, Thomas
AU - Beese, Lorena S.
AU - Kuchta, Robert D.
PY - 2009/6/2
Y1 - 2009/6/2
N2 - 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, N2, N-3, and N6 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 N6 having the most severe impacts. Adding N2 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, N2, N-3, and N6 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-à-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.
AB - 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, N2, N-3, and N6 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 N6 having the most severe impacts. Adding N2 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, N2, N-3, and N6 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-à-vis these other enzymes and, therefore, that nature has evolved at least three mechanistically distinct solutions.
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U2 - 10.1021/bi900104n
DO - 10.1021/bi900104n
M3 - Article
VL - 48
SP - 4633
EP - 4641
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 21
ER -