Purification and characterization of Escherichia coli endonuclease III from the cloned nth gene

The gene which codes for endonuclease III of Escherichia coli has been sequenced. The nth gene was previously subcloned and defined as the gene which led to overproduction of endonuclease III when present on a multicopy plasmid and which created a deficiency in endonuclease III activity when mutated...

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Bibliographic Details
Published in:	Biochemistry (Easton) Vol. 28; no. 10; pp. 4444 - 4449
Main Authors:	Asahara, Hitomi, Wistort, Peter M, Bank, Janet F, Bakerian, Ronald H, Cunningham, Richard P
Format:	Journal Article
Language:	English
Published:	United States American Chemical Society 16-05-1989
Subjects:	Amino Acid Sequence Base Sequence Cloning, Molecular Deoxyribonuclease (Pyrimidine Dimer) DNA, Bacterial - genetics Endodeoxyribonucleases - genetics Endodeoxyribonucleases - isolation & purification endonuclease III Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli Proteins Genes Genes, Bacterial Genetic Vectors Molecular Sequence Data nucleotide sequence
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Summary:	The gene which codes for endonuclease III of Escherichia coli has been sequenced. The nth gene was previously subcloned and defined as the gene which led to overproduction of endonuclease III when present on a multicopy plasmid and which created a deficiency in endonuclease III activity when mutated. The nth gene was sequenced and translated into a predicted polypeptide. The molecular weight (23,546), the amino-terminal amino acid sequence, and the amino acid composition of the polypeptide predicted from the nucleotide sequence are excellent agreement with those same properties determined for the purified protein. Thus, the nth gene is the structural gene for endonuclease III. Inspection of the nucleotide sequence reveals that there is an open reading frame immediately upstream of the nth gene, suggesting that it might be part of an operon. There is a region of dyad symmetry which could form a hairpin stem and loop structure if transcribed into RNA characteristic of a rho-dependent terminator downstream from the nth gene. The nth gene of Escherichia coli has been cloned onto a lambda PL expression vector which yields approximately 300-fold overproduction of endonuclease III. We have purified the enzyme to apparent homogeneity using two chromatographic steps. Our purification scheme allowed the preparation of 117 mg of protein from 190 g of E. coli with a 70% yield. The purified protein has both AP endonuclease activity and DNA N-glycosylase activity. The protein has a Stokes radius of 2.25 nm, a sedimentation coefficient of 2.65 S, a molecular weight of 26,300 in the native state and 27,300 in the denatured state, and a frictional ratio of 1.13.
Bibliography:	istex:D25CDF63C399962247A191126F0E2B507158687F ark:/67375/TPS-GX8SDTQ4-3 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0006-2960 1520-4995
DOI:	10.1021/bi00436a048