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, H309S), which target amino acid residues comprising the abasic DNA endonuclease active site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic site single-stranded RNA (AP-ssRNA) cleavage activity, with concurrent loss of AP site cleavage activity on dsDNA and 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 RNA binding. Interestingly, both endoribonuclease and ssRNA AP site cleavage activity of wild-type APE1 was present in the absence of Mg2+, while ssDNA AP site cleavage required Mg2+ (optimally at 0.5 to 2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA-cleavage by WT APE1[comment: I think I would leave this result out of the abstract), consistent with APE1 leaving a 3′-PO42− group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, AP-ssRNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.
Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in the DNA base excision repair pathway and cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. APE1 also has 3′-5′ DNA exonuclease and 3′ DNA phosphodiesterase activities, and regulates transcription factor DNA binding through its redox regulatory function. The human APE1 has recently been shown to endonucleolytically cleave single-stranded regions of RNA. Towards understanding the biological significance of the endoribonuclease activity of APE1, we examined eight different amino acid substitution variants of APE1 previously identified in the human population. Our study shows that six APE1 variants, D148E, Q51H, I64V, G241R, R237A, and G306A, exhibit a 76–85% reduction in endoribonuclease activity against a specific coding region of the c-myc RNA, yet fully retain the ability to cleave apurinic/apyrimidinic DNA. We found that two APE1 variants, L104R and E126D, exhibit a unique RNase inhibitor-resistant endoribonuclease activity, where the proteins cleave c-myc RNA 3′ of specific single-stranded guanosine residues. Expression of L104R and E126D APE1 variants in bacterial Origami cells leads to a 60–80% reduction in colony formation and a 1.5-fold increase in cell doubling time, whereas the other variants, which exhibit diminished endoribonuclease activity, had no effect. These data indicate that two human APE1 variants exhibit a unique endoribonuclease activity, which correlates with their ability to induce cytotoxicity or slow down growth in bacterial cells and supports the notion of their biological functionality.
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