skip to main content

Title: [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 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:
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]
Research Org:
Harvard Medical School, Boston, MA (United States). Dept. of Biological Chemistry and Molecular Pharmacology
Sponsoring Org:
USDOE, Washington, DC (United States)
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