title: The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA.
abstract: BACKGROUND
  The apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn2+ ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis.
  
  
  METHODS
  The kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions.
  
  
  RESULTS
  We found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage.
  
  
  CONCLUSIONS
  We consider His-83 properly coordinates the active site Zn2+ ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions.
  
  
  CONCLUSIONS
  Our study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk, 630090, Russian Federation.
authors.forename: Elena S
authors.initials: ES
authors.lastname: Dyakonova
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk, 630090, Russian Federation; Novosibirsk State University, Pirogov St., 2, Novosibirsk, 630090, Russian Federation.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Koval
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk, 630090, Russian Federation; Novosibirsk State University, Pirogov St., 2, Novosibirsk, 630090, Russian Federation.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Lomzov
authors.affiliation: Groupe «Réparation de l'ADN», Université Paris-Sud XI, UMR8200 CNRS, Institut Gustave Roussy, Villejuif Cedex F-94805, France.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ishchenko
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk, 630090, Russian Federation; Novosibirsk State University, Pirogov St., 2, Novosibirsk, 630090, Russian Federation. Electronic address: fedorova@niboch.nsc.ru.
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D020134:Catalytic Domain; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
chemical_list: D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D004706:Endodeoxyribonucleases; C083274:Apn1 protein, S cerevisiae; D045643:DNA Repair Enzymes; D015032:Zinc
keywords: 2-Aminopurine; AP endonuclease; Abasic site; Base excision repair; DNA repair; MD simulation