characterization of the endoribonuclease active site of human apurinic/apyrimidinic endonuclease 1
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Wan-Cheol Kim1, BriaDavid M. Wilson III 2 a1Chemistry Program, University oBritish Columbia, Canada V2N 4Z2Laboratory of Molecular Gerontolo251 Bayview Boulevard, Baltimore
Received 26 March 2011;received in revised form
residues contributed primarily to RNA catalysis and not to RNA binding.
doi:10.1016/j.jmb.2011.06.050 J. Mol. Biol. (2011) 411, 960971
Contents lists available at www.sciencedirect.com
Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmbInterestingly, both the endoribonuclease and the ssRNA AP site cleavageactivities ofWTAPE1were present in the absence ofMg2+, while ssDNAAPsite cleavage required Mg2+ (optimally at 0.52.0 mM). We also found that a2-OH on the sugarmoietywas absolutely required for RNA cleavage byWTAPE1, consistent with APE1 leaving a 3-PO4
2 group following cleavage ofRNA. Altogether, our data support the notion that a common active site isshared 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, anobservation that has implications for unraveling the endoribonucleasefunction of APE1 in vivo.
2011 Elsevier Ltd. All rights reserved.27 June 2011;accepted 30 June 2011Available online6 July 2011
Edited by J. Karn
Keywords:endoribonuclease;APE1;RNA*Corresponding author. E-mail addAbbreviations used: APE1, apurin
RNA; WT, wild type; RNase, ribonuCRD, coding region determinant; EMssDNA, single-stranded DNA; EDTNatural Sciences and Engineering R
0022-2836/$ - see front matter 2011 EApyrimidinic Endonuclease 1
n R. Berquist2, Manbir Chohan1, Christopher Uy1,nd Chow H. Lee1f Northern British Columbia, 3333 University Way, Prince George,9gy, National Institute on Aging, National Institutes of Health,, MD 21224, USA
Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalianenzyme in DNA base excision repair that cleaves the DNA phosphodiesterbackbone immediately 5 to abasic sites. Recently, we identified APE1 as anendoribonuclease that cleaves a specific coding region of c-myc mRNA invitro, regulating c-myc mRNA level and half-life in cells. Here, we furthercharacterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNAnuclease activities.Wefound that most site-directed APE1 mutant proteins (N68A, D70A, Y171F,D210N, F266A, D308A, and H309S), which target amino acid residuesconstituting the abasic DNA endonuclease active-site pocket, showedsignificant decreases in endoribonuclease activity. Intriguingly, the D283NAPE1 mutant protein retained endoribonuclease and abasic single-strandedRNA 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 theseCharacterization of the Endoribonuclease Active Site ofress: email@example.com/apyrimidinic endonuclease 1; AP, apurinic/apyrimidinic; ssRNA, single-strandedclease; AP-dsDNA, abasic double-stranded DNA; dsDNA, double-stranded DNA;SA, electrophoretic mobility shift assay; AP-ssDNA, abasic single-stranded RNA;
A, ethylenediaminetetraacetic acid; IDT, Integrated DNA Technologies; NSERC,esearch Council.
lsevier Ltd. All rights reserved.
including the recently discovered single-stranded15
matic function of APE1 resides in the same region(active site) as its other nuclease activities. Further-more, the RNA-cleaving activity of APE1 is active inthe absence of magnesium,32 in stark contrast toabasic double-stranded DNA (AP-dsDNA) endonu-clease activity where magnesium is essential.33
These results provide hints about possible differ-ences in the mechanisms for the RNA-cleaving andAP-dsDNA endonuclease activities of APE1.This study was undertaken to investigate the roles
played by several key DNA nuclease active-siteamino acids in the endoribonuclease activity ofAPE1, with the goal of understanding the catalyticmechanism for RNA cleavage by APE1. Our resultsshow that many, but not all, amino acid residuescritical for AP-dsDNA incision are also importantfor the endoribonuclease activity of APE1. Ourresults have implications for understanding the
Fig. 1. AP-dsDNA endonuclease activity of the struc-tural mutants of APE1. (a) The AP-dsDNA endonucleaseactivities of WT APE1 and APE1 structural mutants wereassessed as described in Materials and Methods. Recom-binant proteins (0.14 nM; lanes 210) were incubated with5--32P-radiolabeled AP-dsDNA. The 18-nt AP-dsDNAsubstrates and the 9-nt incised products are shown witharrows. (b) The structure and sequence of the 18-nt AP-dsDNA substrate strand and the 9-nt single-strandedproduct are shown. The 18-mer oligonucleotide containsthe model analog of an abasic site, tetrahydrofuran (F).and abasic single-stranded31 RNA-cleaving activitiesof APE1.Introduction
There is increasing evidence that endonucleolyticcleavage of mRNA plays a critical role in eukaryoticand mammalian gene expression.14 Recent reportson several enzymes possessing endoribonucleolyticactivities have highlighted their roles in mediatingthe posttranscriptional regulation of geneexpression.59 Endoribonucleases that cleavemRNA appear to be induced by stress signals,leading to profound effects on cell growth anddisease development due to their abilities to controlrelevant transcript levels.4,10 For instance, ribonu-clease (RNase) L becomes activated by 2,5-linkedoligoadenylates (25A) after interferon signaling inresponse to viral infection.11 RNase L activation, inturn, destabilizes mRNAs of ribosomal proteins andthe RNA-binding protein HuR.12,13 Similarly, poly-somal ribonuclease-1 regulates -globin mRNAupon phosphorylation by an activated c-Src.14
We have recently identified apurinic/apyrimidinicendonuclease 1 (APE1) as an endoribonuclease thatcleaves c-myc mRNA in vitro.15 We further showedthat APE1 can regulate c-myc mRNA level and half-life in cells, possibly via this endoribonucleaseactivity. APE1 is a multifunctional protein withroles in DNA base excision repair and redoxactivation of DNA-binding transcription factors.16
The functional regions for the DNA repair and redoxfunctions of APE1 seem to be independent of eachother.17 The abasic DNA endonuclease domain of thehuman protein consists of several important aminoacids between Asn68 and His309, as determined byX-crystallography18 and functional studies.1923 Onthe other hand, the N-terminal region of APE1harbors the redox center, which consists of criticalcysteine residues (Cys65 and Cys93) important foractivating various transcription factors implicated inapoptosis and cell growth (e.g., AP1, Egr-1, NF-B,p53, c-Jun, and HIF).16,24 APE1 has been found topossess multiple DNA nuclease functions, including3 phosphodiesterase,25 35 exonuclease,26 and 3phosphatase activities.25,27,28 In addition, RNase-H-type activity of APE1 has been reported.29 Studiesindicate that the exonuclease and abasic DNAendonuclease activities share a common active site,with both activities being sensitive to mutations incritical amino acids such as Glu96, Asp210, andHis309.30 The fact that most, if not all, of its nucleaseactivities share a common active-site center makes itchallenging to study the contribution and signifi-cance of each nuclease activity separately in cells,
Endoribonuclease Active Site of Human APE1We have previously shown that H309N and E96Amutants of APE1 have no RNA-cleaving activity,suggesting that the domain containing this enzy-Contr
1 2 3 4 5 6 7 8 9 10
18 nt9 nt
961structural basis of RNA cleavage by APE1, as wellas for dissecting the significance of the endoribonu-clease function of APE1 in vivo.
APE1 active-site residues participate in bothAP-dsDNA incision and endoribonucleaseactivities
Recombinant wild-type (WT) APE1 and APE1structural mutants containing a single site-specificamino acid change in the active site previouslyshown to be important for AP-dsDNA endonucleaseactivity (N68A, D70A, Y171F, D210N, F266A,D283N, D308A, and H309S) were purified to nearhomogeneity and analyzed for purity on an SDSpolyacrylamide gel, as described previously.21 Thedouble-stranded DNA (dsDNA) apurinic/apyrimi-
intensity of the decay products 1742CA, 1747UA,and 1751UA. All of the mutants exhibited abrogatedendoribonuclease activity, with the notable excep-tion of the D283N mutant (Fig. 2b and Table 1).These results showed that most of the amino acidresidues important for AP-dsDNA incision are alsocritical for the endoribonuclease activity of APE1.
Most APE1 active-site residues are notindividually critical for c-myc CRD RNA binding
The reduced endoribonuclease activity of thestructural mutants reported in Fig. 2b could be dueto their reduced ability to bind RNA and/or adeficiency in catalysis. To evaluate the first premise,we assayed the ability of these structural mutants to
962 Endoribonuclease Active Site of Human APE1dinic (AP) site cleavage activity of these APE1structural mutants was first assessed using apreviously employed 18 -bp AP-dsDNA substrate34
(Fig. 1). AP-dsDNA endonuc