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Title: In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

Abstract

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.

Authors:
;
Publication Date:
Research Org.:
Dept. of Microbiology, Univ. of Florida Medical School, Gainesville, FL (US)
OSTI Identifier:
6829915
Resource Type:
Journal Article
Journal Name:
Mol. Cell. Biol.; (United States)
Additional Journal Information:
Journal Volume: 8:6
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADENOVIRUS; DNA SEQUENCING; DNA-CLONING; ENDONUCLEASES; FRACTIONATION; RECOMBINANT DNA; CELL TRANSFORMATIONS; ENZYME ACTIVITY; HELA CELLS; IN VITRO; MOLECULAR STRUCTURE; TISSUE CULTURES; CLONING; DNA; DNA-ASE; ENZYMES; ESTERASES; HYDROLASES; MICROORGANISMS; NUCLEIC ACIDS; ONCOGENIC VIRUSES; ORGANIC COMPOUNDS; PARASITES; PHOSPHODIESTERASES; SEPARATION PROCESSES; STRUCTURAL CHEMICAL ANALYSIS; VIRUSES; 550200* - Biochemistry

Citation Formats

Gottlieb, J, and Muzyczka, N. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences. United States: N. p., 1988. Web. doi:10.1128/MCB.8.6.2513.
Gottlieb, J, & Muzyczka, N. In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences. United States. doi:10.1128/MCB.8.6.2513.
Gottlieb, J, and Muzyczka, N. Wed . "In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences". United States. doi:10.1128/MCB.8.6.2513.
@article{osti_6829915,
title = {In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences},
author = {Gottlieb, J and Muzyczka, N},
abstractNote = {When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat and in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.},
doi = {10.1128/MCB.8.6.2513},
journal = {Mol. Cell. Biol.; (United States)},
number = ,
volume = 8:6,
place = {United States},
year = {1988},
month = {6}
}