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

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and Expression of the cDNA Encoding the Human Homologue of the DNA Repair Enzyme, Escherichia coli Endonuclease III

“…Although purified E. coli endonuclease III has a characteristic absorption peak at 410 nm and might be expected to appear blue in solution, the color of solutions containing approximately 0.5 mg/ml or greater of purified endonuclease III are typically yellow-brown (19). Similarly, a solution of the purified GST fusion protein at similar concentrations of protein was also yellow, while a solution of the simultaneously purified non-fusion GST protein was colorless.…”

“…Purification of E. coli Endonuclease III-Endonuclease III was purified from E. coli strain UC6444 carrying the plasmid pHIT1 as described previously (19).…”

DNA glycosylases in the base excision repair of DNA

“…Overproduction of EndoIII protects against lethal effects of ionizing radiation and chemical oxidants [180], and the protein is considered to be a prime defence mechanism against oxidized pyrimidines in E. coli. EndoIII has been purified to physical homogeneity [181] and its crystal structure has been solved [160,162]. The 23.4 kDa protein (211 amino acids) is elongated and bilobal, with a central cleft separating a continuous six-α-helix barrel domain and a four-α-helix domain formed by the Nterminal helix and three C-terminal helices.…”

Investigation of systemic folate status, impact of alcohol intake and levels of DNA damage in mononuclear cells of breast cancer patients

“…In the modified comet assays, T cells embedded on slides were treated with either formamidopyrimidine glycosylase (FPG), which recognises oxidatively modified purines (Boiteux et al, 1992), or with endonuclease III (ENDO III), which recognises oxidatively modified pyrimidines (Asahara et al, 1989). These enzymes nick DNA at the sites of oxidatively damaged nucleotides, creating single-strand breaks which can be detected with the alkaline comet assay.…”