A Unique Region in Bacteriophage T7 DNA Polymerase Important for Exonucleolytic Hydrolysis of DNA

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) ⌬144 -157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol ⌬144 -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 ⌬144 -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.Bacteriophage T7-encoded gene 5 protein is a replicative DNA polymerase that also has an associated 3Ј-5Ј exonuclease activity that is active on both single-stranded (ss) 1 and doublestranded (ds) DNA (1). By itself, the 80-kDa gene 5 protein (gp5) is distributive for DNA synthesis and catalyzes the addition of less than 15 nucleotides before dissociating from a primer-template terminus (2). Association in a 1:1 complex with the 12-kDa Escherichia coli host protein thioredoxin increases the processivity of polymerization by gp5; the gp5⅐thioredoxin complex catalyzes the addition of thousands of nucleotides per cycle of polymerization at a rate of 300 nucleotides per second (3, 4). The association with thioredoxin also increases the exonuclease activity of gp5 on dsDNA several hundred-fold but has little effect on the hydrolysis of ssDNA (2). The complex of gp5 and thioredoxin will be referred to as T7 DNA polymerase in this report.T7 DNA polymerase, like other members of the E. coli DNA polymerase I (Pol I) family, has a bipartite architecture with distinct C-terminal polymerase and N-terminal exonuclease domains (5). A 2.2-Å resolution crystal structure of T7 DNA polymerase in complex with a primer-template and an incoming dideoxynucleoside triphosphate was solved in the polymerization mode (see Fig. 1). This structure captured the polymerase in the act of adding a nucleotide to a growing chain of DNA. The polymerase domain is likened to a right hand composed of thumb, fingers, and palm subdomains that converge to form a DNA binding groove that leads to the polymerase active site. The processivity factor, E. coli thioredoxin, is bound to the thumb subdomain. In this structure, there are no visible contacts between the exonuclease domain and DNA. The exonuclease and polymerase active sites a...

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…158 More recently, the para-nitrophenol ester of deoxythymidine 5′-monophosphate (pNP-dTMP) has been used as substrate to directly monitor spectrophotometrically the formation of the product of the hydrolytic reaction, paranitrophenol. 189,225 In general, it has been found that the rates of hydrolysis of pNP-TMP by various exonucleases are significantly lower than those determined for the hydrolysis of polynucleotide substrates. For example, k cat values reported for the hydrolysis of pNP-TMP and single-stranded DNA by the subunit of DNA Pol III are 0.32 s -1 (at 25°C and pH ) 8.0) and 200 s -1 (pH ) 8.0), respectively.…”

“…189,224 Similar trends have been observed for the 3′-5′ exonuclease domains of E. coli DNA Pol I and T7 DNA polymerase. 225,226 A region (residues 144-157) in T7 DNA Polymerase has been identified as being crucial for DNA binding during catalysis. While the deletion of this region does not affect the 3′-5′ exonuclease active site, it leads to a substantial decrease of the hydrolytic activity in the presence of DNA substrates, indicating that substrate binding may trigger conformational changes required for optimal catalytic efficiency.…”

“…While the deletion of this region does not affect the 3′-5′ exonuclease active site, it leads to a substantial decrease of the hydrolytic activity in the presence of DNA substrates, indicating that substrate binding may trigger conformational changes required for optimal catalytic efficiency. 225 A majority of 3′-5′ exonucleases have optimal activity in alkaline environments with pH optima ranging from 7.5 to 10.2. 158,189,216 The pH dependence of the K m values of the subunit of E. coli Pol III for the substrate pN-dTMP associates a basic amino acid with a pK a >8.5 in substrate interactions (possibly Arg159).…”

Dual Role of φ29 DNA Polymerase Lys529 in Stabilisation of the DNA Priming-Terminus and the Terminal Protein-Priming Residue at the Polymerisation Site

“…Hydrolysis was studied by monitoring p -nitrophenol production at 420 nm with a Hitachi U-200 spectophotomer at 25°C, essentially as described [19]. Production of p -nitrophenol was plotted against time and adjusted to a rectangular hyperbola by least-squares non-linear regression, using Kaleidagraph 3.6.4. software.…”

Functional Importance of Bacteriophage ϕ29 DNA Polymerase Residue Tyr148 in Primer-terminus Stabilisation at the 3′-5′ Exonuclease Active Site

“…Hydrolysis was studied by monitoring p-nitrophenol production at 420 nm with a Hitachi U-2000 spectrophotometer at 25°C, as described. 42 Production of p-nitrophenol was plotted against time and adjusted to a rectangular hyperbola by least-squares non-linear regression, using the Kaleidagraph 3.6.4 software. Linearity in the production of p-nitrophenol was obtained in the time range 5-100 s. Slopes obtained by linear regression adjustments of those points allowed us to calculate the activity for the hydrolysis of the phosphoester bond (s − 1 ).…”