Cleavage of Single- and Double-Stranded DNAs Containing an Abasic Residue by Escherichia Coli Exonuclease III (AP Endonuclease VI)

Abstract

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.

Characterization of the AP Endonucleases fromThermoplasma volcaniumandLactobacillus plantarum: Contributions of Two Important Tryptophan Residues to AP Site Recognition

“…2B, products 2) are generated by exonucleolytic cleavage of the AP endonucleolytic product from the 3 0 end to the 5 0 side. 23,24) In TvoExo and LplExo, a band corresponding to product 1 was detected. This phenomenon clearly indicates that TvoExo and LplExo have AP endonuclease activities.…”

“…Moreover, it is obvious that TvoExo and LplExo have 3 0 -5 0 exonuclease activities, judging from the ladder-like pattern products. Each relative amount of the AP endonucleolytic product at 23 C was calculated from the total amount of all products per amount of the initial substrate. TvoExo and LplExo showed similar AP endonucleolytic cleavage efficiencies (106% and 78% respectively) to ExoIII.…”

“…The preheated proteins were incubated with dsDNA, containing an AP site, at 23 C for 1 min. The reaction products were detected using 7 M urea denaturing 20% polyacrylamide gels.…”

Systematic identification of synthetic lethal mutations with reduced-genome <i>Escherichia coli</i>: synthetic genetic interactions among <i>yoaA</i>, <i>xthA</i> and <i>holC</i> related to survival from MMS exposure

“…The Δ21a strain was constructed by introducing a large-scale chromosome deletion, LD3-17-1 (deletion location is 1,821,337-1,860,378 bp and the length is 39,042 bp; for further information, see the PEC database, http://www.shigen.nig.ac.jp/ ecoli/pec/), into Δ20a, suggesting that a second gene responsible for synthetic lethality is present in the chromosomal region affected by this deletion (Supplementary Tables S1 and S4). We suspected xthA as the responsible gene in this region: it encodes DNA endonuclease VI/ DNA exonuclease III, and is also involved in DNA repair (Mol et al, 1995;Shida et al, 1996). XthA is a major AP endonuclease that removes damaged DNA at cytosines and guanines by cleaving on the 3'-side of an AP site via a beta-elimination reaction.…”

Gold Nanoparticle-Based Colorimetric Sensors for Detection of DNA and Small Molecules

“…The Exo III then recognized the newly-formed 3'-hydroxyl termini on the nicked ends of the probe fragments and rapidly catalyzed the stepwise removal of 5'-mononucleotides through its exonucleolytic activity. 270,286 This ultimately sheared the probe fragments from the particle but released the PM strand intact. The released PM target was then recycled and hybridized with another probe DNA on the same or a different AuNP to begin the cycle anew.…”