S-glutathionylation of cysteine 99 in the APE1 protein impairs abasic endonuclease activity.
Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a central participant in the base excision repair pathway, exhibiting AP endonuclease activity that incises the DNA backbone 5' to an abasic site. Besides its prominent role as a DNA repair enzyme, APE1 was separately identified as a protein called redox effector factor 1, which is able to enhance the DNA binding activity of several transcription factors through a thiol-exchange-based reduction-oxidation mechanism. In the present study, we found that human APE1 is S-glutathionylated under conditions of oxidative stress both in the presence of glutathione in vitro and in cells. S-glutathionylated APE1 displayed significantly reduced AP endonuclease activity on abasic-site-containing oligonucleotide substrates, a result stemming from impaired DNA binding capacity. The combination of site-directed mutagenesis, biochemical assays, and mass spectrometric analysis identified Cys99 in human APE1 as the critical residue for the S-glutathionylation that leads to reduced AP endonuclease activity. This modification is reversible by reducing agents, which restore APE1 incision function. Our studies describe a novel posttranslational modification of APE1 that regulates the DNA repair function of the protein.
Laboratory of Molecular Gerontology, Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA.
Yun-Jeong
YJ
Kim
Daemyung
D
Kim
Jennifer L
JL
Illuzzi
Sarah
S
Delaplane
Dian
D
Su
Michel
M
Bernier
Michael L
ML
Gross
Millie M
MM
Georgiadis
David M
DM
Wilson
D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011499:Protein Processing, Post-Translational; D019163:Reducing Agents / Q000494:pharmacology
D019163:Reducing Agents; D006861:Hydrogen Peroxide; D004720:Endonucleases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005978:Glutathione; D003545:Cysteine