title: Characterization of two independent amino acid substitutions that disrupt the DNA repair functions of the yeast Apn1.
abstract: The members of the Endo IV family of DNA repair enzymes, including Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV, possess the capacity to cleave abasic sites and to remove 3'-blocking groups at single-strand breaks via apurinic/apyrimidinic (AP) endonuclease and 3'-diesterase activities, respectively. In addition, Endo IV family members are able to recognize and incise oxidative base damages on the 5'-side of such lesions. We previously identified eight amino acid substitutions that prevent E. coli endonuclease IV from repairing damaged DNA in vivo. Two of these substitutions were glycine replacements of Glu145 and Asp179. Both Glu145 and Asp179 are among nine amino acid residues within the active site pocket of endonuclease IV that coordinate the position of a trinuclear Zn cluster required for efficient phosphodiester bond cleavage. We now report the first structure-function analysis of the eukaryotic counterpart of endonuclease IV, yeast Apn1. We show that glycine substitutions at the corresponding conserved amino acid residues of yeast Apn1, i.e., Glu158 and Asp192, abolish the biological function of this enzyme. However, these Apn1 variants do not exhibit the same characteristics as the corresponding E. coli mutants. Indeed, the Apn1 Glu158Gly mutant, but not the E. coli endonuclease IV Glu145Gly mutant, is able to bind DNA. Moreover, Apn1 Asp192Gly completely lacks enzymatic activity, while the activity of the E. coli counterpart Asp179Gly is reduced by approximately 40-fold. The data suggest that although yeast Apn1 and E. coli endonuclease IV exhibit a high degree of structural and functional similarity, differences exist within the active site pockets of these two enzymes.
authors.affiliation: Guy-Bernier Research Centre, University of Montreal, 5415 de l'Assomption, Montreal, Quebec, Canada H1T 2M4.
authors.forename: Arshad
authors.initials: A
authors.lastname: Jilani
authors.forename: Ratsavarinh
authors.initials: R
authors.lastname: Vongsamphanh
authors.forename: Anick
authors.initials: A
authors.lastname: Leduc
authors.forename: Laurent
authors.initials: L
authors.lastname: Gros
authors.forename: Murat
authors.initials: M
authors.lastname: Saparbaev
authors.forename: Dindial
authors.initials: D
authors.lastname: Ramotar
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D045643:DNA Repair Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000378:metabolism / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*
chemical_list: D000596:Amino Acids; D008164:Luminescent Proteins; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D004706:Endodeoxyribonucleases; C083274:Apn1 protein, S cerevisiae; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D045643:DNA Repair Enzymes; D010100:Oxygen