Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing
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 activity 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.
- 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:
- 5545844
- Report Number(s):
- DOE/ER/60688-T1; ON: DE91015445
- Country of Publication:
- United States
- Language:
- English
Similar Records
[Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing]: Progress report
Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993
Related Subjects
DNA POLYMERASES
PROTEIN ENGINEERING
STRUCTURE-ACTIVITY RELATIONSHIPS
DNA SEQUENCING
AIDS VIRUS
AMINO ACID SEQUENCE
BACTERIOPHAGES
DNA HELICASES
DNA REPLICATION
DNA-ASE
ELECTROPHORESIS
ESCHERICHIA COLI
MAGNESIUM IONS
MANGANESE IONS
MOLECULAR BIOLOGY
MUTAGENESIS
PROGRESS REPORT
PROTEIN STRUCTURE
PYROPHOSPHATES
RNA PROCESSING
RNA-ASE
BACTERIA
CHARGED PARTICLES
DOCUMENT TYPES
ENZYMES
ESTERASES
HYDROLASES
IONS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEIC ACID REPLICATION
NUCLEOTIDYLTRANSFERASES
OXYGEN COMPOUNDS
PARASITES
PHOSPHODIESTERASES
PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VIRUSES
550200* - Biochemistry
550400 - Genetics