THE PURIFICATION AND CHARACTERIZATION OF THE X-RAY ENDONUCLEASE OF ESCHERICHIA COLI (ENZYMOLOGY).
Item
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Title
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THE PURIFICATION AND CHARACTERIZATION OF THE X-RAY ENDONUCLEASE OF ESCHERICHIA COLI (ENZYMOLOGY).
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Identifier
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AAI8501146
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identifier
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8501146
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Creator
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KATCHER, HAROLD LAWRENCE.
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Contributor
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Susan S. Wallace
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Date
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1984
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, General
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Abstract
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This work concerns the purification and characterization of the X-ray endonuclease of E. coli. The X-ray endonuclease was first detected as an activity present in crude lysates of Escherichia coli that nicked X-irradiated DNA in a dose-dependent manner (Strniste and Wallace, 1975). The X-ray endonuclease was purified by chromatography on DNA-agarose, Sephadex gel filtration, hydroxylapatite chromatography, and phosphocellulose chromatography. Several thousand fold purification was obtained. Parallel assays on modified DNA and oligonucleotide substances established that the X-ray endonuclease was active on DNA containing 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. Glycerol gradient centrifugation showed two peaks of activity at positions corresponding to 25,000 daltons and 13,000 daltons which indicated that the X-ray endonuclease may be composed of two similar or identical subunits.;Analysis of DNA substrates following X-ray endonuclease treatment showed that the X-ray endonuclease nicked at the 3' side of a base lesion to yield 3'OH and 5'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. Further studies showed that the thymine glycol-DNA glycosylase activity was NEM sensitive. NEM was also shown to inhibit endonuclease activity on UV-irradiated DNA, X-irradiated DNA, and urea-containing DNA.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biology
