title: Functional variants of human APE1 rescue the DNA repair defects of the yeast AP endonuclease/3'-diesterase-deficient strain.
abstract: Human APE1 is an essential enzyme performing functions in DNA repair and transcription. It possesses four distinct repair activities acting on a variety of base and sugar derived DNA lesions. APE1 has seven cysteine residues and Cys65, and to a lesser extent Cys93 and Cys99, is uniquely involved in maintaining a subset of transcription factors in the reduced and active state. Four of the cysteines Cys93, 99, 208 and 310 of APE1 are located proximal to its active site residues Glu96, Asp210 and His309 involved in processing damaged DNA, raising the possibility that missense mutation of these cysteines could alter the enzyme DNA repair functions. An earlier report documented that serine substitution of the individual cysteine residues did not affect APE1 ability to cleave an abasic site oligonucleotide substrate in vitro, except for Cys99Ser, although any consequences of these variants in the repair of in vivo DNA lesions were not tested. Herein, we mutated all seven cysteines of APE1, either singly or in combination, to alanine and show that none of the resulting variants interfered with the enzyme DNA repair functions. Cross-specie complementation analysis reveals that these APE1 cysteine variants fully rescued the yeast DNA repair deficient strain YW778, lacking AP endonucleases and 3'-diesterases, from toxicities caused by DNA damaging agents. Moreover, the elevated spontaneous mutations arising in strain YW778 from the lack of the DNA repair activities were completely suppressed by the APE1 cysteine variants. These findings suggest that the cysteine residues of APE1 are unlikely to play a role in the DNA repair functions of the enzyme in vivo. We also examine other APE1 missense mutations and provide the first evidence that the variant Asp308Ala with normal AP endonuclease, but devoid of 3'→5' exonuclease, displays hypersensitivity to the anticancer drug bleomycin, and not to other agents, suggesting that it has a defect in processing unique DNA lesions. Molecular modeling reveals that Asp308Ala cannot make proper contact with Mg(2+) and may alter the enzyme ability to cleave or disassociate from specific DNA lesions.
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4.
authors.forename: Emily
authors.initials: E
authors.lastname: Ayoub
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4.
authors.forename: Abdelghani
authors.initials: A
authors.lastname: Mazouzi
authors.affiliation: Groupe Réparation de l'ADN, Université Paris Sud, Laboratoire Stabilité Génétique et Oncogenèse CNRS, UMR 8200, Gustave-Roussy Cancer Center, F-94805 Villejuif Cedex, France; SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Ave. , Novosibirsk 630090, Russia.
authors.forename: Inga
authors.initials: I
authors.lastname: Grin
authors.affiliation: Groupe Réparation de l'ADN, Université Paris Sud, Laboratoire Stabilité Génétique et Oncogenèse CNRS, UMR 8200, Gustave-Roussy Cancer Center, F-94805 Villejuif Cedex, France.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ishchenko
authors.affiliation: Research Institute of the McGill University Health Centre and Department of Medicine, McGill University, 1650 Cedar Avenue, Montreal, QC H3G 1A4, Canada.
authors.forename: Jinjiang
authors.initials: J
authors.lastname: Fan
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Yang
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4.
authors.forename: Taramatti
authors.initials: T
authors.lastname: Harihar
authors.affiliation: Groupe Réparation de l'ADN, Université Paris Sud, Laboratoire Stabilité Génétique et Oncogenèse CNRS, UMR 8200, Gustave-Roussy Cancer Center, F-94805 Villejuif Cedex, France.
authors.forename: Murat
authors.initials: M
authors.lastname: Saparbaev
authors.affiliation: Maisonneuve-Rosemont Hospital, Research Center, Université de Montréal 5415 Boul. de l' Assomption, Montréal, Québec, Canada H1T 2M4. Electronic address: dindial.ramotar@umontreal.ca.
authors.forename: Dindial
authors.initials: D
authors.lastname: Ramotar
mesh_terms: D003545:Cysteine / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D003545:Cysteine
keywords: Base-excision repair; DNA repair; Human AP endonuclease; Mutations; Oxidative DNA damage; Yeast