(Characterization and modification of Phage T7 DNA polymerase for use in DNA sequencing): Progress report
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 for 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.
- Research Organization:
- Harvard Medical School, Boston, MA (USA)
- DOE Contract Number:
- FG02-88ER60688
- OSTI ID:
- 6606270
- Report Number(s):
- DOE/ER/60688-1; ON: DE89004119
- Resource Relation:
- Other Information: Portions of this document are illegible in microfiche products
- Country of Publication:
- United States
- Language:
- English
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[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. Final report, June 1, 1988--January 31, 1996
Related Subjects
DNA SEQUENCING
MODIFICATIONS
ESCHERICHIA COLI
DNA POLYMERASES
BACTERIOPHAGES
EVALUATION
GENE REGULATION
INACTIVATION
MUTAGENESIS
PROGRESS REPORT
TRANSCRIPTION
BACTERIA
DOCUMENT TYPES
ENZYMES
MICROORGANISMS
NUCLEOTIDYLTRANSFERASES
PARASITES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VIRUSES
550200* - Biochemistry