Purification of a novel UV-endonuclease from Escherichia coli
Ultraviolet (UV) irradiation produces a variety of damages to purine residues in DNA, which include photoalkylated purines, purine-pyrimidine photoadducts and adenine photodimers. In this study, an enzymatic activity which incises UV-irradiated DNA at sites of adenines was isolated from an Escherichia coli mutant. This endonuclease activity was purified 86-fold with a 28% yield by ion-exchange and affinity chromatography. Using polynucleotides radiolabeled by nick translation, the substrate specificity of purine photoproduct endonuclease was determined. UV-irradiated poly(dA)-poly(dT) was the only substrate for the purine photoproduct endonuclease. The enzyme specifically released radiolabeled material from the purine strand of the poly(dA)-poly(dT). DNA sequence analysis showed that the site of cleavage of UV-irradiated DNA by the purified enzyme was in regions of multiple adjacent adenine residues. The purine photoproduct endonuclease did not incise the substrate at regions of the DNA in which pyrimidine photoproducts could be formed. The in vivo formation of adenine-containing photoproducts in cultured human cells was demonstrated using the purine photoproduct endonuclease as a probe. The number of enzyme sensitive sites increased with increasing irradiation doses. It was also shown that adenine photoproducts were removed in vivo from the cellular DNA. The lesions were rapidly excised from UV-irradiated cells; the half-life of the adenine-containing photoproducts was approximately 1.5 hrs and remaining enzyme-sensitive sites were removed from the cells within 4 hrs. This purified enzymatic activity may be utilized to isolate the enzyme-released product for subsequent characterization. In addition, using the enzyme as a probe, the action spectrum for the induction of the adenine photoproduct can be determined to assess the biological significance of these UV-induced lesions.
- Research Organization:
- West Virginia Univ., Morgantown, WV (United States)
- OSTI ID:
- 7201133
- Resource Relation:
- Other Information: Thesis (Ph.D.)
- Country of Publication:
- United States
- Language:
- English
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63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
DNA
BIOLOGICAL RADIATION EFFECTS
STRAND BREAKS
ENDONUCLEASES
PURIFICATION
SUBSTRATES
ADENINES
ENZYME ACTIVITY
ESCHERICHIA COLI
PURINES
ULTRAVIOLET RADIATION
AMINES
ANTIMETABOLITES
AROMATICS
AZAARENES
BACTERIA
BIOLOGICAL EFFECTS
DNA-ASE
DRUGS
ELECTROMAGNETIC RADIATION
ENZYMES
ESTERASES
HETEROCYCLIC COMPOUNDS
HYDROLASES
MICROORGANISMS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
PROTEINS
RADIATION EFFECTS
RADIATIONS
550200* - Biochemistry
560120 - Radiation Effects on Biochemicals
Cells
& Tissue Culture