Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases

Abstract

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.

A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents

“…The bacterial expression system for producing wild-type recombinant human APE1 protein (i.e., pETApe) was generated as described (51). Overlapping PCR mutagenesis was used (see refs.…”

“…28, 52) to sequentially introduce two codon changes (i.e., the E96Q and D210N amino acid substitutions) into the pETApe construct to create the double mutant APE1 protein, ED. ED was expressed and purified from the recombinant pET-ED plasmid as detailed for the wild-type APE1 protein (51).…”

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease☆

“…Mammalian AP endo, initially isolated from human tissue (19,26,34) and cell lines (4,21,22), was confirmed as the E. coli equivalent because of its enzymatic activity and because the gene complemented E. coli deficient in exonuclease III for resistance to alkylating agents (9,40,42). AP endo binds abasic (AP) site-containing DNA in the absence of divalent cation with remarkable affinity as shown using an electrophoretic mobility shift assay (EMSA) (11,28,29,43,47) and single turnover (ST) kinetics (43). As a processive enzyme (5), AP endo also binds DNA lacking an AP-site with surprising affinity (2,43).…”

“…In this paper we have used two methodologies to generate a more complete picture of how AP endo recognizes DNA with and without an APsite. EMSA is routinely employed to measure DNA binding with proteins that interact with a particular consensus sequence (15,18,39,46,49,50) or with a particular lesion (11,14,29,47), while single turnover and steady state kinetics examine binding from the viewpoint of catalytic activity.…”

Synthesis and fluorescence study of 7-azaindole in DNA oligonucleotides replacing a purine base

“…There are mainly two groups of 'bases' synthesized and incorporated into DNA oligomers: those made by minor modification of the natural bases or the derivatives of cyclic aromatic compounds. While the former includes 2-aminopurine [1][2][3][4][5][6][7][8], inosine [9] and isoinosine [9][10][11], 3-and 7-deazaadenine [12][13][14][15], and 3-and 7-deazaguanine [14,16], the latter includes analogs of pteridine [17][18][19] and indole [20][21][22][23][24], benzene [23][24][25][26], naphthalene and pyrene derivatives [24], The first group of compounds, when replacing a natural base in duplex DNA, can still form hydrogen bonds with the base in the opposite strand, while the second group of compounds cannot form any hydrogen bonds.…”