Properties of SV40 large T antigen involved in the replication of SV40 DNA
The role of phosphorylation in regulating the biochemical properties of SV40 large T antigen (Tag) was analyzed. Treatment of purified Tag with calf intestinal alkaline phosphatase (CIAP) removed 80% of the {sup 32}P label. CIAP treated Tag displayed an increased ability to support SV40 DNA replication in vitro. This paralleled an activation of specific DNA binding to site II, an essential element within the SV40 origin of replication (ori). The ATPase activity of dephosphorylated Tag remained unchanged. To evaluate the properties of Tag devoid of mammalian post translational modifications, a bacterial expression system was used. Purified E. coli Tag did not bind to site II, and therefore could not unwind ori containing plasmids or efficiently replicate SV40 DNA in vitro. However, E. coli Tag functioned as a helicase, and bound to DNA fragments containing either site I or the wild type (wt) ori in a manner identical to mammalian Tag. A single DNA binding domain is responsible for specific DNA binding to either of two different sites embedded in the SV40 ori, and the intrinsic preference of full length Tag for either site I, or site II appears to be modulated by phosphorylation. Several Tag point mutants were examined in a series of assays which reflect partial biochemical reactions required for the in vitro replication of SV40 DNA.
- Research Organization:
- State Univ. of New York, Stony Brook, NY (USA)
- OSTI ID:
- 6759804
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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ENZYME ACTIVITY
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SIMIAN VIRUS
MOLECULAR BIOLOGY
CALVES
ESCHERICHIA COLI
INTESTINES
PHOSPHORUS 32
PLASMIDS
TRACER TECHNIQUES
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CATTLE
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENZYMES
GASTROINTESTINAL TRACT
GENES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MICROORGANISMS
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ODD-ODD NUCLEI
ORGANS
PARASITES
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RUMINANTS
VERTEBRATES
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550201* - Biochemistry- Tracer Techniques