Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing

Technical Report · Tue Jan 01 04:00:00 EST 1991

OSTI ID:5545844

Richardson, C C

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: DOE; 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

Related Subjects

550200* -- Biochemistry
550400 -- Genetics
59 BASIC BIOLOGICAL SCIENCES
AIDS VIRUS
AMINO ACID SEQUENCE
BACTERIA
BACTERIOPHAGES
CHARGED PARTICLES
DNA HELICASES
DNA POLYMERASES
DNA REPLICATION
DNA SEQUENCING
DNA-ASE
DOCUMENT TYPES
ELECTROPHORESIS
ENZYMES
ESCHERICHIA COLI
ESTERASES
HYDROLASES
IONS
MAGNESIUM IONS
MANGANESE IONS
MICROORGANISMS
MOLECULAR BIOLOGY
MOLECULAR STRUCTURE
MUTAGENESIS
NUCLEIC ACID REPLICATION
NUCLEOTIDYLTRANSFERASES
OXYGEN COMPOUNDS
PARASITES
PHOSPHODIESTERASES
PHOSPHORUS COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PROGRESS REPORT
PROTEIN ENGINEERING
PROTEIN STRUCTURE
PYROPHOSPHATES
RNA PROCESSING
RNA-ASE
STRUCTURAL CHEMICAL ANALYSIS
STRUCTURE-ACTIVITY RELATIONSHIPS
TRANSFERASES
VIRUSES