Excision of C-4′-oxidized Deoxyribose Lesions from Double-stranded DNA by Human Apurinic/Apyrimidinic Endonuclease (Ape1 Protein) and DNA Polymerase β

Abstract

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase ␤, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase ␤ excised the 5-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase ␤-mediated excision of 5-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3-phosphoglycolate esters.

Role of 2-Phosphoglycolate Phosphatase ofEscherichia coliin Metabolism of the 2-Phosphoglycolate Formed in DNA Repair

“…A large number of enzymes that repair these termini have been reported for bacteria (8,31), yeasts (30), and higher eukaryotes (38).…”

Expression profile of genes coding for DNA repair in human oocytes using pangenomic microarrays, with a special focus on ROS linked decays

“…The same conclusion is reached irrespective of the methodology used to examine DNA integrity: sperm DNA fragmentation and decays related to reactive oxygen species (ROS) lead to reduced fertility [2,3], and ROS have a heavily deleterious impact on sperm DNA [4,5]. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidic (AP) sites [6,7], but 8 oxo deoxyguanosine (8 OXO dG or 8 OH dG) can also be produced. We recently described the formation of ethenoadenosine and ethenoguanosine (1,N 2 -etheno-2′-deoxyguanosine (ɛdGuo), and 1,N 6 -etheno-2′-deoxyadenosine (ɛdAdo) in human sperm [8].…”

“…Shortened telomeres, in relation to age and sperm DNA fragmentation [9] may lead to inappropriate end to end fusion and the formation of chromosome bridges. On the other hand, paternal DNA always carries a varying level of DNA strand breaks [1][2][3][4][5][6][7][8][9][10]. These decays are the result of ROS and/or anomalies of topoisomerase activity during spermatogenesis.…”

The Saccharomyces cerevisiae ETH1 Gene, an Inducible Homolog of Exonuclease III That Provides Resistance to DNA-Damaging Agents and Limits Spontaneous Mutagenesis

“…DNA polymerases may stall during replication upon encountering an AP site (27) or, for some DNA and RNA polymerases, may bypass the AP site (19). AP endonucleases also participate in the repair of DNA strand break damage caused by ionizing agents, hydrogen peroxide, and radiomimetic agents such as bleomycin (7,55).Base excision repair is an essential biochemical pathway. Deletion of the gene encoding the major AP endonuclease, DNA polymerase ␤, or XRCC1 protein in a mouse by targeted homologous recombination results in the absence of viable homozygous mice (embryonic lethal phenotype [25]).…”

“…DNA polymerases may stall during replication upon encountering an AP site (27) or, for some DNA and RNA polymerases, may bypass the AP site (19). AP endonucleases also participate in the repair of DNA strand break damage caused by ionizing agents, hydrogen peroxide, and radiomimetic agents such as bleomycin (7,55).…”