The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease, ARP is involved in the plant nucleotide incision repair pathway.
Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3'→5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (kcat/KM=134 and 7.3 μM-1·min-1, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3'→5' exonuclease activities (kcat/KM=314 and 34 μM-1·min-1, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp-/-mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H2O2, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.
Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, 0530040, Almaty, Kazakhstan.
Zhiger
Z
Akishev
Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, 0530040, Almaty, Kazakhstan.
Sabira
S
Taipakova
Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, 0530040, Almaty, Kazakhstan.
Botagoz
B
Joldybayeva
Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France.
Caroline
C
Zutterling
Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, 0530040, Almaty, Kazakhstan.
Izat
I
Smekenov
Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France.
Alexander A
AA
Ishchenko
Novosibirsk State University, Novosibirsk 630090, Russia; SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia.
Dmitry O
DO
Zharkov
Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, 0530040, Almaty, Kazakhstan. Electronic address: Amangeldy.Bisenbaev@kaznu.kz.
Amangeldy K
AK
Bissenbaev
Groupe «Réparation de l'ADN», Equipe Labellisée par la Ligue Nationale contre le Cancer, CNRS UMR8200, Université Paris-Sud, Gustave Roussy Cancer Campus, F-94805 Villejuif Cedex, France. Electronic address: murat.saparbaev@gustaveroussy.fr.
Murat
M
Saparbaev
D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D003839:Deoxyadenosines / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D035264:Flowers / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008958:Models, Molecular; D018384:Oxidative Stress; D030821:Plants, Genetically Modified; D017433:Protein Structure, Secondary; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D029681:Arabidopsis Proteins; D003839:Deoxyadenosines; C010922:5,6-dihydrouridine; D020122:tert-Butylhydroperoxide; D008741:Methyl Methanesulfonate; D006861:Hydrogen Peroxide; C540281:Ape1L protein, Arabidopsis; C540282:Ape2 protein, Arabidopsis; C540283:Arp protein, Arabidopsis; D004720:Endonucleases; C058118:2'-deoxyadenosine; D014529:Uridine
3'-repair phosphodiesterase; AP endonuclease; DNA repair; alkylating agent; apurinic/apyrimidinic sites; base excision repair; hydrogen peroxide; nucleotide incision repair; organic peroxide; oxidative DNA damage