title: Characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1.
abstract: Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5' to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg(2+), while ssDNA AP site cleavage required Mg(2+) (optimally at 0.5-2.0 mM). We also found that a 2'-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3'-PO(4)(2-) group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.
authors.affiliation: Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George, British Columbia, Canada V2N 4Z9.
authors.forename: Wan-Cheol
authors.initials: WC
authors.lastname: Kim
authors.forename: Brian R
authors.initials: BR
authors.lastname: Berquist
authors.forename: Manbir
authors.initials: M
authors.lastname: Chohan
authors.forename: Christopher
authors.initials: C
authors.lastname: Uy
authors.forename: David M
authors.initials: DM
authors.lastname: Wilson
authors.forename: Chow H
authors.initials: CH
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004722:Endoribonucleases / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C489427:MYC protein, human; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA; D004722:Endoribonucleases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase