Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product.
We investigated the interaction dynamics of human abasic endonuclease, the Ape1 protein (also called Ref1, Hap1, or Apex), with its DNA substrate and incised product using electrophoretic assays and site-specific amino acid substitutions. Changing aspartate 283 to alanine (D283A) left 10% residual activity, contrary to a previous report, but complementation of repair-deficient bacteria by the D283A Ape1 protein was consistent with its activity in vitro. The D308A, D283/D308A double mutant, and histidine 309 to asparagine proteins had 22, 1, and approximately 0. 02% of wild-type Ape1 activity, respectively. Despite this range of enzymatic activities, all the mutant proteins had near-wild-type binding affinity specific for DNA containing a synthetic abasic site. Thus, substrate recognition and cleavage are genetically separable steps. Both the wild-type and mutant Ape1 proteins bound strongly to the enzyme incision product, an incised abasic site, which suggested that Ape1 might exhibit product inhibition. The use of human DNA polymerase beta to increase Ape1 activity by eliminating the incision product supports this conclusion. Notably, the complexes of the D283A, D308A, and D283A/D308A double mutant proteins with both intact and incised abasic DNA were significantly more stable than complexes containing wild-type Ape1, which may contribute to the lower turnover numbers of the mutant enzymes. Wild-type Ape1 protein bound tightly to DNA containing a one-nucleotide gap but not to DNA with a nick, consistent with the proposal that substrate recognition by Ape1 involves a space bracketed by duplex DNA, rather than mere flexibility of the DNA.
Masuda
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Y
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Bennett
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RA
Demple
B
B
D019943:Amino Acid Substitution; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004247:DNA / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D004247:DNA; D019951:DNA Polymerase beta; D004720:Endonucleases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase