Abstract
A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E. coli DNA polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 polymerase (pol) Delta144-157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol Delta144-157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol Delta144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3'-5' exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase. Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.
Highlights
Bacteriophage T7-encoded gene 5 protein is a replicative DNA polymerase that has an associated 3Ј-5Ј exonuclease activity that is active on both single-stranded1 and doublestranded DNA [1]
One approach to understanding the molecular basis for these differences in activities is to compare T7 DNA polymerase and Klenow fragment for unique differences. Such an approach has been used successfully to identify a loop in the DNA binding crevice that interacts with the DNA primase domain of T7 gene 4 protein [8], a unique segment in the thumb subdomain of T7 gene 5 protein that binds thioredoxin [6], and a single amino acid residue in the active site that accounts for the discrimination against dideoxynucleotides [7]
While the efficiencies of plating of T7⌬5 phage on E. coli C600 cells expressing wild-type T7 DNA polymerase or T7 pol ⌬144 –157 are similar, plaques formed by T7 pol ⌬144 – 157 are smaller
Summary
Bacteriophage T7-encoded gene 5 protein is a replicative DNA polymerase that has an associated 3Ј-5Ј exonuclease activity that is active on both single-stranded (ss) and doublestranded (ds) DNA [1]. One approach to understanding the molecular basis for these differences in activities is to compare T7 DNA polymerase and Klenow fragment for unique differences Such an approach has been used successfully to identify a loop in the DNA binding crevice that interacts with the DNA primase domain of T7 gene 4 protein [8], a unique segment in the thumb subdomain of T7 gene 5 protein that binds thioredoxin [6], and a single amino acid residue in the active site that accounts for the discrimination against dideoxynucleotides [7].
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