Abstract
We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9670-9675) by substituting an oligonucleotide containing random sequences for codons 659-671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature- sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29-68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZα forward mutation assay, A661E and T664R yielded mutation frequencies at least 7- and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis.
| Original language | English |
|---|---|
| Pages (from-to) | 11228-11235 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 272 |
| Issue number | 17 |
| DOIs | |
| Publication status | Published - 25-04-1997 |
| Externally published | Yes |
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All Science Journal Classification (ASJC) codes
- Biochemistry
- Molecular Biology
- Cell Biology
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Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I. / Suzuki, Motoshi; Avicola, Amy K.; Hood, Leroy; Loeb, Lawrence A.
In: Journal of Biological Chemistry, Vol. 272, No. 17, 25.04.1997, p. 11228-11235.Research output: Contribution to journal › Article
TY - JOUR
T1 - Low fidelity mutants in the O-helix of Thermus aquaticus DNA polymerase I
AU - Suzuki, Motoshi
AU - Avicola, Amy K.
AU - Hood, Leroy
AU - Loeb, Lawrence A.
PY - 1997/4/25
Y1 - 1997/4/25
N2 - We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9670-9675) by substituting an oligonucleotide containing random sequences for codons 659-671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature- sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29-68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZα forward mutation assay, A661E and T664R yielded mutation frequencies at least 7- and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis.
AB - We screened 67 mutants in the O-helix of Thermus aquaticus (Taq) DNA polymerase I (pol I) for altered fidelity of DNA synthesis. These mutants were obtained (Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9670-9675) by substituting an oligonucleotide containing random sequences for codons 659-671, and selecting for complementation of a growth defect in Escherichia coli caused by temperature- sensitive host pol I. Thirteen mutants decreased fidelity in a screen that employed primer extension reactions lacking one of four complementary deoxynucleoside triphosphates (dNTPs). Three mutants were purified and exhibited 29-68% of wild-type specific activity. Homogeneous polymerases A661E, A661P, and T664R extended primers further than the wild-type, synthesizing past template nucleotides for which the complementary dNTP was absent. The data indicate that both misinsertion of incorrect nucleotides and extension of mispaired primer termini were increased. In a lacZα forward mutation assay, A661E and T664R yielded mutation frequencies at least 7- and 25-fold greater, respectively, than that of the wild-type polymerase. These findings emphasize the importance of the O-helix in substrate recognition and are compatible with a role for pyrophosphate release in enhancing fidelity of DNA synthesis.
UR - http://www.scopus.com/inward/record.url?scp=0030897085&partnerID=8YFLogxK
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U2 - 10.1074/jbc.272.17.11228
DO - 10.1074/jbc.272.17.11228
M3 - Article
VL - 272
SP - 11228
EP - 11235
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -