Discrimination between right and wrong purine dNTPs by DNA polymerase I from Bacillus stearothermophilus.
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.
Department of Chemistry and Biochemistry, University of Colorado, UCB 215, Boulder, Colorado 80309, USA.
Michael
M
Trostler
Alison
A
Delier
Jeff
J
Beckman
Milan
M
Urban
Jennifer N
JN
Patro
Thomas E
TE
Spratt
Lorena S
LS
Beese
Robert D
RD
Kuchta
D004256:DNA Polymerase I / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D009584:Nitrogen / Q000378:metabolism; D011685:Purine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D003838:Deoxyadenine Nucleotides; D003854:Deoxyribonucleotides; D011685:Purine Nucleotides; D004256:DNA Polymerase I; C026600:2'-deoxyadenosine triphosphate; D009584:Nitrogen