[Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report
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.
- Research Organization:
- Harvard Medical School, Boston, MA (United States). Dept. of Biological Chemistry and Molecular Pharmacology
- Sponsoring Organization:
- USDOE; USDOE, Washington, DC (United States)
- DOE Contract Number:
- FG02-88ER60688
- OSTI ID:
- 6854160
- Report Number(s):
- DOE/ER/60688-5; ON: DE93006149
- Country of Publication:
- United States
- Language:
- English
Similar Records
Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing. Final report, June 1, 1988--January 31, 1996
Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing
Related Subjects
DNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
PROTEIN STRUCTURE
DNA SEQUENCING
GENE MUTATIONS
PROGRESS REPORT
PROTEIN ENGINEERING
DOCUMENT TYPES
ENZYMES
KINETICS
MUTATIONS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROTEINS
REACTION KINETICS
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550200* - Biochemistry
550400 - Genetics