A cell-free system for DNA repair synthesis using purified enzymes from the Novikoff hepatoma
Novikoff DNA polymerase-{beta} and Novikoff DNase V have been used in a cell-free DNA excision repair system for UV-irradiated substrates to determine their DNA repair capabilities. The repair system was shown to depend upon UV-irradiated DNA, incision by phage T4 UV-endonuclease, excision by DNase V and synthesis by DNA polymerase-{beta}; ligation was not included. Highly purified calf thymus DNA was UV-irradiated at 500-750 J/m{sup 2} and incised by T4 UV-endonuclease. The repair system was used to follow the purification of DNase V and DNA polymerase-{beta}. For increased specificity, the parameters of UV-irradiation, incision, excision and synthesis were confirmed on highly supercoiled, covalently closed, phage PM2 DNA. Optimal DNA and Mg{sup 2+} concentrations were determined for the repair assay, which was shown to be linear with respect to time. Excision of the 3{prime}-apyrimidinic site and the 5{prime}-pyrimidine dimer by bidirectional DNase V, presumed to occur from the above experiments, was studied more thoroughly using lightly UV-irradiated ({sup 3}H)poly(dT)poly (dA), labeled in both the base and the sugar, and incised with T4 UV-endonuclease.
- Research Organization:
- Cincinnati Univ., OH (USA)
- OSTI ID:
- 7001308
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
DNA
GENETIC RADIATION EFFECTS
DNA REPAIR
BACTERIOPHAGES
CALVES
DNA POLYMERASES
PYRIMIDINE DIMERS
THYMUS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
ANIMALS
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
BODY
CATTLE
DOMESTIC ANIMALS
ELECTROMAGNETIC RADIATION
ENZYMES
GENETIC EFFECTS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
LYMPHATIC SYSTEM
MAMMALS
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANS
PARASITES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
RUMINANTS
TRANSFERASES
VERTEBRATES
VIRUSES
560130* - Radiation Effects on Microorganisms
550201 - Biochemistry- Tracer Techniques