Characterization of the Endoribonuclease Active Site of Human Apurinic/Apyrimidinic Endonuclease 1

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<ul><li><p>Human Apurinic/</p><p>Wan-Cheol Kim1, BriaDavid M. Wilson III 2 a1Chemistry Program, University oBritish Columbia, Canada V2N 4Z2Laboratory of Molecular Gerontolo251 Bayview Boulevard, Baltimore</p><p>Received 26 March 2011;received in revised form</p><p>residues contributed primarily to RNA catalysis and not to RNA binding.</p><p>doi:10.1016/j.jmb.2011.06.050 J. Mol. Biol. (2011) 411, 960971</p><p>Contents lists available at www.sciencedirect.com</p><p>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</p><p>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.</p><p> 2011 Elsevier Ltd. All rights reserved.27 June 2011;accepted 30 June 2011Available online6 July 2011</p><p>Edited by J. Karn</p><p>Keywords:endoribonuclease;APE1;RNA*Corresponding author. E-mail addAbbreviations used: APE1, apurin</p><p>RNA; WT, wild type; RNase, ribonuCRD, coding region determinant; EMssDNA, single-stranded DNA; EDTNatural Sciences and Engineering R</p><p>0022-2836/$ - see front matter 2011 EApyrimidinic Endonuclease 1</p><p>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</p><p>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: leec@unbc.ca.ic/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;</p><p>A, ethylenediaminetetraacetic acid; IDT, Integrated DNA Technologies; NSERC,esearch Council.</p><p>lsevier Ltd. All rights reserved.</p></li><li><p>including the recently discovered single-stranded15</p><p>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</p><p>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</p><p>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</p><p>5 3</p><p>AP-dsDNA</p><p>G</p><p>C</p><p>G</p><p>A</p><p>TC</p><p>CA</p><p>TG</p><p>18nt</p><p>5</p><p>C</p><p>CA</p><p>TG</p><p>9ntProduct</p><p>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</p><p>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</p><p>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</p><p>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,</p><p>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</p><p>ol</p><p>WT N68A</p><p>D70A</p><p>Y171</p><p>FD2</p><p>10NF2</p><p>66A</p><p>D283</p><p>NH3</p><p>09S</p><p>D308</p><p>A</p><p>1 2 3 4 5 6 7 8 9 10</p><p>(a)</p><p>(b)</p><p>CG</p><p>C</p><p>GC</p><p>A</p><p>G</p><p>C</p><p>A</p><p>T</p><p>G</p><p>C</p><p>A</p><p>TGC</p><p>AT</p><p>F</p><p>C</p><p>A</p><p>T</p><p>G</p><p>A</p><p>TG</p><p>3 5</p><p>C</p><p>GTG</p><p>18 nt9 nt</p><p>961structural basis of RNA cleavage by APE1, as wellas for dissecting the significance of the endoribonu-clease function of APE1 in vivo.</p></li><li><p>Results</p><p>APE1 active-site residues participate in bothAP-dsDNA incision and endoribonucleaseactivities</p><p>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-</p><p>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.</p><p>Most APE1 active-site residues are notindividually critical for c-myc CRD RNA binding</p><p>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</p><p>leas</p><p>FoAP</p><p>co</p><p>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</p><p>(Fig. 1). AP-dsDNA endonuclease activity wasessentially abolished in N68A, Y171F, D210N,D283N, and H309S mutants, whereas significantreduction in activity was seen with D70A, F266A,and D308A mutants. The reduction in AP-dsDNAendonuclease activities observed with these APE1structural mutants correlated well with previousstudies (Table 1)1923 indicating the importance ofthese residues in AP-dsDNA incision activity. TheAP-dsDNA endonuclease results are shown in Fig.1a, and the quantitative summary is reported inTable 1. AP-dsDNA endonuclease activities werecalculated by quantifying the ratio of the density ofthe product to that of the substrate+ product.We next assessed the same panel of APE1</p><p>structural mutants for their ability to cleave 5-32P-labeled c-myc coding region determinant (CRD)RNA substrate (Fig. 2a). To quantitatively comparethe endoribonuclease activity of APE1 structuralmutants to that of WT APE1, we measured the</p><p>Table 1. Summary of fold reduction in the endoribonucmutants as compared to WT APE1</p><p>APE1 structuralmutants</p><p>Fold reductionin AP-dsDNA incisiona in</p><p>N68A 60023</p><p>D70A UDd</p><p>E96A 600100,00021,30</p><p>Y171F 500021</p><p>D210N 25,00021</p><p>N212A UC19</p><p>F266A 620</p><p>D283N 1022</p><p>D308A 52521,33</p><p>H309N 100,00030</p><p>H309S UD</p><p>AB, abolished; UD, undetermined; and UC, unchanged activity asa Information obtained from the literature.b Data obtained from Fig. 1a and replicates.c Data obtained from Fig. 2b and replicates.d For the D70R mutant, the fold reduction was 27-fold.23bind c-myc CRD RNA by electrophoretic mobilityshift assay (EMSA).Experiments were performed initially to optimize</p><p>the binding activity of WT APE1 on c-myc CRDRNA. We found that varying the pH from 6.0 to 7.4or adding detergents such as Triton X-100 had noeffect, while the addition of heparin completelyabolished the binding of APE1 to c-myc CRD RNA(data not shown). We then assayed WT APE1 for itsability to bind c-mycCRDRNA. At 706 and 1412 nM,a slower-migrating RNAAPE1 complex was ob-servable as a smear (lanes 2 and 3; Fig. 3a), while atconcentrations above 1412 nM, APE1 appeared tobind all of the RNA substrates, resulting in theformation of a single RNAAPE1 complex (lanes 46; Fig. 3a). A saturation binding curve was generatedfrom replicate experiments using WT APE1 concen-trations from 0 to 1412 nM (Fig. 3b). Fitting thesaturation binding data using the Hill equation, wefound that the dissociation constant Kd of WT APE1binding c-myc CRD RNAwas 880+113 nM. The Hillcoefficient was determined to be 1.0, suggesting thatone molecule of APE1 binds one molecule of c-myc</p><p>e and AP-dsDNA incision activities of APE1 structural</p><p>ld reduction-dsDNA incisionb</p><p>Fold reduction in endoribonucleaseactivity against c-myc CRDc</p><p>AB AB6.7 ABUD AB15</p><p>AB ABAB ABUD UD30.4 ABAB UC1.4 ABUD AB15</p><p>AB UD</p><p>mpared to that of WT APE1.</p></li><li><p>WTtro</p><p>l</p><p>2Con</p><p>1</p><p>(b)(a)</p><p>1742CA</p><p>1747UA1751UA</p><p>1757UA</p><p>1766CA1768CA</p><p>1771CA1773UA</p><p>GUU</p><p>AG</p><p>UC</p><p>UG</p><p>AA</p><p>AC</p><p>A</p><p>AAA</p><p>A</p><p>A</p><p>C</p><p>GAC</p><p>A</p><p>CGG</p><p>GCC</p><p>CCC</p><p>C</p><p>AAAA</p><p>AA</p><p>UA</p><p>G U UAU</p><p>1710</p><p>1720</p><p>1730</p><p>1740</p><p>1750</p><p>1760</p><p>1770</p><p>1780</p><p>1790</p><p>AGG C</p><p>CU</p><p>G</p><p>U</p><p>A U</p><p>A</p><p>C</p><p>A</p><p>GG C</p><p>C</p><p>AA U</p><p>U</p><p>1775CA</p><p>1727CA</p><p>1730UG</p><p>Endoribonuclease Active Site of Human APE1CRD RNA. Our findings on the progressive shift ofthe APE1RNA complex with increasing concentra-tions of APE1 have similarly been reported using a58-nt single-stranded RNA (ssRNA),29 and this mayreflect the on- and off-binding rates of the protein(i.e., the instability of the complex). Interestingly, theKd of APE1 binding to c-myc CRD RNA is similar tothat of the RNA-binding protein CRD-BP, a proteinpreviously shown to specifically bind c-myc CRDRNA.35 A number of studies have shown that the Kdof APE1 binding to DNA substrates is in the range of0.87125 nM,20,3638 suggesting that APE1 has alower affinity for RNA substrates.We next evaluated the relative ability of the panel</p><p>of APE1 structural mutants to bind c-mycCRDRNA.We employed protein concentrations thatwere in thelinear range for binding to the RNA substrate byWTAPE1 (01412 nM) (Fig. 3a and b). We reasoned thatif there are differences in binding affinity among themutant proteins, theywouldmost likely be observedin this concentration range. The left panel in Fig. 3cshows that both WT APE1 (lanes 24) and E96A(lanes 68) proteins exhibit similar binding affinitiesfor c-myc CRD RNA, with 5060% of c-myc RNAbound. In contrast, H309N (lanes 1012) showedreduced binding, with 2035% of c-myc RNA bound.</p><p>c-myc 1705-1792 CRD RNAA C C A G A U C C U C C A A G C</p><p>GC</p><p>CG</p><p>5 3</p><p>Fig. 2. Structural mutants of APE1 display reduced endsequence of c-myc 17051792 CRD are shown. Arrows indicatewas carried out on c-myc CRD RNA with APE1 and its struRecombinant proteins (1.4 M; lanes 210) were tested againtotal reaction volume of 20 l for 25 min at 37 C. Numbers o1757UA1751UA1747UA1742CA</p><p>1730UG1727CA</p><p>1775CA1773UA</p><p>1768CA1771CA</p><p>N68A</p><p>D70A</p><p>Y171</p><p>FD2</p><p>10NF2</p><p>66A</p><p>D283</p><p>NH3</p><p>09S</p><p>D308</p><p>A</p><p>3 4 5 6 7 8 9 10</p><p>963We found that N68A (lanes 1820), Y171F (lanes 2224), and D210N (lanes 2628) all displayed RNAbinding affinities comparable toWTAPE1 (lanes 24and 1416). Interestingly, at lower concentrations(353 and 706 nM; lanes 30 and 31), the D283Nmutant protein showed a modest decrease inbinding, with 3040% of c-myc RNA bound. At ahigher concentration (1412 nM; lane 32), the RNAbinding affinity of D283N became comparable tothat of WT APE1 (lanes 24 and 1416; Fig. 3c).</p><p>APE1 D283N active-site mutation abolishesabasic single-stranded DNA cleavage activitybut retains abasic single-stranded RNAcleavage activity</p><p>Mutation at D283 has been consistently shown tonegatively affect the AP-dsDNA endonuclease ac-tivity of APE1 (Fig. 1a).22,33,39,40 Our results above,however, indicate that...</p></li></ul>

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