The role of His-83 of yeast apurinic/apyrimidinic endonuclease Apn1 in catalytic incision of abasic sites in DNA

• Published in: Biochimica et biophysica acta Vol. 1850; no. 6; pp. 1297 - 1309
• Main Authors: Dyakonova, Elena S., Koval, Vladimir V., Lomzov, Alexander A., Ishchenko, Alexander A., Fedorova, Olga S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2015
• Subjects: 2-Aminopurine; Abasic site; active sites; Amino Acid Sequence; amino acids; antimalarials; AP endonuclease; Base excision repair; Catalysis; catalytic activity; Catalytic Domain; crystal structure; DNA; DNA damage; DNA Repair; DNA Repair Enzymes - chemistry; DNA Repair Enzymes - genetics; DNA Repair Enzymes - metabolism; DNA, Fungal - chemistry; DNA, Fungal - metabolism; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; enzyme activity; Escherichia coli; fluorescence; guidelines; Histidine; ions; Kinetics; mammals; MD simulation; Molecular Dynamics Simulation; Molecular Sequence Data; mutants; Mutation; Nucleic Acid Conformation; Protein Conformation; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; yeasts; zinc; Zinc - metabolism; Abasic site; MD simulation; 2-Aminopurine; F; MD; ODN; AP endonuclease; 2-aPu; DNA repair; Base excision repair; BER; AP
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2015.03.001

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. 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. 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. 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. 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. •Kinetics of abasic DNA incision by mutant endonuclease H83A Apn1 was studied.•Substituting His-83 with Ala in Apn1 decreases substrate binding and incision.•2-aPu residue upstream of the abasic site arrests the incision activity of H83A Apn1.•MD simulations of H83A Apn1 structure revealed Zn2+ content reduced in active site.•Adding Zn2+ ions in the reaction mixture restores H83A Apn1 incision activity.