Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.
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.
Erzberger
J P
JP
Biology and Biotechnology Research Program, L-452, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA.
Barsky
D
D
Schärer
O D
OD
Colvin
M E
ME
Wilson
D M
DM
D000409:Alanine / Q000378:metabolism; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009692:Nucleic Acid Heteroduplexes; D010649:Phenylalanine / Q000378:metabolism; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D010649:Phenylalanine; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D000409:Alanine