A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides
- Harvard Medical School, Boston, MA (United States)
Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four dideoxynucleotides by >2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis. 29 refs., 4 figs., 2 tabs.
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-88ER60688
- OSTI ID:
- 96601
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 14 Vol. 92; ISSN PNASA6; ISSN 0027-8424
- Country of Publication:
- United States
- Language:
- English
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