Purification and characterization of the x-ray endonuclease of Escherichia coli

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1984

OSTI ID:7009575

Katcher, H L

This work concerns the purification and characterization of the x-ray endonuclease of E. coli. The x-ray endonuclease was purified by chromatography on DNA-agarose, Sephadex gel filtration, hydroxylapatite chromatography, and phosphocellulose chromatography. Parallel assays on modified DNA and oligonucleotide substances established that the x-ray endonuclease was active on DNA contain in apurinic and apyrimidinic sites, thymine glycol and urea residues, and undefined lesions produced by UV and X radiation. Characterization of the x-ray endonuclease by gel filtration gave a molecular weight of about 25,000 dalton while SDS-polyacrylamide gel electrophoresis of the most purified preparations showed a single band corresponding to a molecular weight of about 13,000 daltons. Analysis of DNA substrates following x-ray endonuclease treatment showed that the x-ray endonuclease nicked at the 3{prime} side of a base lesion to yield 3{prime}OH and 5{prime}PO termini. Analysis of the acid/alcohol soluble products of the digestion of specifically modified synthetic poly dT:dA by the x-ray endonuclease showed this enzyme to have DNA glycosylase activities that released both thymine glycol and urea residues from DNA. Inhibitor studies showed the thymine-glycol endonuclease activity was inhibited by NEM while the AP endonuclease was not. NEM was also shown to inhibit endonuclease activity on UV-irradiated DNA, X-irradiated DNA, and urea-containing DNA.

• Research Organization: City Univ. of New York, NY (USA)
• OSTI ID: 7009575
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
560120 -- Radiation Effects on Biochemicals
Cells
& Tissue Culture
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMIDES
ANTIMITOTIC DRUGS
BACTERIA
BIOLOGICAL EFFECTS
BIOLOGICAL RADIATION EFFECTS
CARBONIC ACID DERIVATIVES
CHROMATOGRAPHY
DNA
DRUGS
ELECTROMAGNETIC RADIATION
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
EXTREME ULTRAVIOLET RADIATION
HYDROLASES
IMIDES
IONIZING RADIATIONS
LIQUID COLUMN CHROMATOGRAPHY
MICROORGANISMS
MOLECULAR STRUCTURE
MOLECULAR WEIGHT
NEM
NUCLEASES
NUCLEIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
PURIFICATION
RADIATION EFFECTS
RADIATIONS
RADIOSENSITIZERS
SEPARATION PROCESSES
ULTRAVIOLET RADIATION
UREA
X RADIATION