title: Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii.
abstract: DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis.
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, 46202, United States.
authors.forename: David O
authors.initials: DO
authors.lastname: Onyango
authors.forename: Arunasalam
authors.initials: A
authors.lastname: Naguleswaran
authors.forename: Sarah
authors.initials: S
authors.lastname: Delaplane
authors.forename: April
authors.initials: A
authors.lastname: Reed
authors.forename: Mark R
authors.initials: MR
authors.lastname: Kelley
authors.forename: Millie M
authors.initials: MM
authors.lastname: Georgiadis
authors.forename: William J
authors.initials: WJ
authors.lastname: Sullivan
mesh_terms: D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics / Q000378:metabolism*; D043223:Deoxyribonuclease IV (Phage T4-Induced) / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D010802:Phylogeny; D021381:Protein Transport; D016415:Sequence Alignment; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics
chemical_list: D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043223:Deoxyribonuclease IV (Phage T4-Induced); D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase