A cell-free system for DNA repair synthesis using purified enzymes from the Novikoff hepatoma

Thesis/Dissertation · Fri Jan 01 04:00:00 EST 1988

OSTI ID:7001308

Small, P K

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
• Country of Publication: United States
• Language: English

Related Subjects

550201 -- Biochemistry-- Tracer Techniques
560130* -- Radiation Effects on Microorganisms
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
ANIMALS
BACTERIOPHAGES
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
BIOLOGICAL RECOVERY
BIOLOGICAL REPAIR
BODY
CALVES
CATTLE
DNA
DNA POLYMERASES
DNA REPAIR
DOMESTIC ANIMALS
ELECTROMAGNETIC RADIATION
ENZYMES
GENETIC EFFECTS
GENETIC RADIATION EFFECTS
HYDROGEN COMPOUNDS
ISOTOPE APPLICATIONS
LYMPHATIC SYSTEM
MAMMALS
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOTIDYLTRANSFERASES
ORGANIC COMPOUNDS
ORGANS
PARASITES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PYRIMIDINE DIMERS
RADIATION EFFECTS
RADIATIONS
RECOVERY
REPAIR
RUMINANTS
THYMUS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
ULTRAVIOLET RADIATION
VERTEBRATES
VIRUSES