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Title: [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report

Abstract

This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4more » protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.« less

Publication Date:
Research Org.:
Harvard Medical School, Boston, MA (United States). Dept. of Biological Chemistry and Molecular Pharmacology
Sponsoring Org.:
USDOE, Washington, DC (United States)
OSTI Identifier:
10111697
Report Number(s):
DOE/ER/60688-5
ON: DE93006149
DOE Contract Number:
FG02-88ER60688
Resource Type:
Technical Report
Resource Relation:
Other Information: PBD: [1992]
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; DNA POLYMERASES; BIOCHEMICAL REACTION KINETICS; PROTEIN STRUCTURE; DNA SEQUENCING; GENE MUTATIONS; PROTEIN ENGINEERING; PROGRESS REPORT; 550200; 550400; BIOCHEMISTRY; GENETICS

Citation Formats

Not Available. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report. United States: N. p., 1992. Web. doi:10.2172/10111697.
Not Available. [Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report. United States. doi:10.2172/10111697.
Not Available. Thu . "[Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report". United States. doi:10.2172/10111697. https://www.osti.gov/servlets/purl/10111697.
@article{osti_10111697,
title = {[Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report},
author = {Not Available},
abstractNote = {This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3` to 5` exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNA sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.},
doi = {10.2172/10111697},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Thu Dec 31 00:00:00 EST 1992},
month = {Thu Dec 31 00:00:00 EST 1992}
}

Technical Report:

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  • The overall goal of this project is to understand in molecular terms the complex process by which a DNA polymerase and its accessory proteins faithfully copy a DNA template. Such knowledge will provide us with a reasonable approach to modifying these proteins by chemical and genetic procedures to increase their usefulness with DNA sequence analysis. We have chosen the DNA polymerase of bacteriophage T7 as a model system. During the next period we will focus on several topics. In order to improve the yield and purity of the T7 gene 5 protein/thioredoxin complex we hope to coexpress the genes formore » both proteins in the same cell and to improve the purification procedure. The physico-chemical studies proposed include crystallization of gene 5 protein. In order to understand the molecular mechanism by which thioredoxin confers processivity on the polymerization reaction we will examine the interaction of the proteins with each other and with well defined primer-templates. A major effort will be directed to obtaining mutationally altered proteins that affect these interactions. Detailed studies will elucidate the precise mechanism by which active oxygen species specifically inactivate the exonuclease active site of gene 5 protein. A major effort will be directed toward determining the location of the metal binding site, the identity and location of the damaged residues, and the location of both the exonuclease and polymerase active sites. Such information will be used to genetically modify the gene 5 protein and thioredoxin to eliminate undesirable properties such as discrimination against nucleotide analogues and to enhance other properties. To identify a single amino acid residue essential for the exonuclease activity of the gene 5 protein. To date we have successfully inactivated the exonuclease activity by deleting 28 amino acids from the gene 5 protein.« less
  • This project focuses on the DNA polymerase and accessory proteins of phage T7 for use in DNA sequence analysis. T7 DNA polymerase (gene 5 protein) interacts with accessory proteins for the acquisition of properties such as processivity that are necessary for DNA replication. One goal is to understand these interactions in order to modify the proteins to increase their usefulness with DNA sequence analysis. Using a genetically modified gene 5 protein lacking 3' to 5' exonuclease activity we have found that in the presence of manganese there is no discrimination against dideoxynucleotides, a property that enables novel approaches to DNAmore » sequencing using automated technology. Pyrophosphorolysis can create problems in DNA sequence determination, a problem that can be eliminated by the addition of pyrophosphatase. Crystals of the gene 5 protein/thioredoxin complex have now been obtained and X-ray diffraction analysis will be undertaken once their quality has been improved. Amino acid changes in gene 5 protein have been identified that alter its interaction with thioredoxin. Characterization of these proteins should help determine how thioredoxin confers processivity on polymerization. We have characterized the 17 DNA binding protein, the gene 2.5 protein, and shown that it interacts with gene 5 protein and gene 4 protein. The gene 2.5 protein mediates homologous base pairing and strand uptake. Gene 5.5 protein interacts with E. coli Hl protein and affects gene expression. Biochemical and genetic studies on the T7 56-kDa gene 4 protein, the helicase, are focused on its physical interaction with T7 DNA polymerase and the mechanism by which the hydrolysis of nucleoside triphosphates fuels its unidirectional translocation on DNA.« less
  • This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less
  • This project focuses on the phage T7 DNA polymerase (gene 5 protein) and its accessory proteins for use in DNA sequence analysis. Crystallization of the gene 5 protein/thioredoxin complex is underway. We have genetically modified the gene 5 protein to eliminate its exonuclease activity. In the presence of Mn{sup 2+} ions there is no discrimination against dideoxynucleoside triphosphates by T7 DNA polymerase, a property that enables novel approaches to DNA sequencing. We have modified the polymerization reaction using other proteins: the gene 4 proteins, gene 2.5 protein, and gene 5.5 protein. The 56 kDa gene 4 protein catalyzes helicase activitymore » but is devoid of primase activity. The 63 kDa genes 4 protein protein has both activities. The gene 2.5 protein binds to single-stranded DNA and catalyzes homologous base-pairing and strand uptake into duplex DNA. The gene 2.5 protein physically interacts with both the gene 5 protein and the gene 5 protein. We have analyzed synthesis catalyzed by reverse transcriptase from HIV-1 and characterized the processing of the RNA primer for (+) strand DNA synthesis. 9 refs.« less
  • This project has focused on the DNA polymerase of phage T7 for use in DNA sequencing. A complex of T7 DNA polymerase and E. coli thioredoxin form a highly processive DNA polymerase. The exonuclease activity of the enzyme can be reduced by chemical or genetic modifications resulting in an enzyme that has several properties useful in sequencing including high processivity and lack of discrimination against dideoxynucleotides. Manganese ion eliminates all discrimination against ddNTPs allowing sequence determination based on band intensity. A single tyrosine residue in the active site of T7 DNA polymerase is responsible for the efficient incorporation of ddNMPs.more » Replacement of the phenylalanine at this position in Klenow or Taq DNA polymerase with tyrosine eliminates discrimination against ddNTPs, a property that has advantages for cycle sequencing. Pyrophosphorolysis catalyzed by a polymerase results in the hydrolysis of specific fragments in DNA sequencing reactions, a problem that is eliminated by the addition of pyrophosphatase. The thioredoxin domain of gene 5 protein has been identified and transferred to Klenow DNA polymerase to make it processive. We have crystallized a complex of T7 DNA polymerase/thioredoxin bound to a primer-template in the presence of a dNTP.« less