doi:10.1016/j.jmb.2011.06.050 J. Mol. Biol. (2011) 411, 960–971

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

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

Wan-Cheol Kim1, Brian R. Berquist2, Manbir Chohan1, Christopher Uy1,David M. Wilson III 2 and Chow H. Lee1⁎1Chemistry Program, University of Northern British Columbia, 3333 University Way, Prince George,British Columbia, Canada V2N 4Z92Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health,251 Bayview Boulevard, Baltimore, MD 21224, USA

Received 26 March 2011;received in revised form27 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 E

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 theseresidues contributed primarily to RNA catalysis and not to RNA binding.Interestingly, both the endoribonuclease and the ssRNA AP site cleavageactivities ofWTAPE1were present in the absence ofMg2+, while ssDNAAPsite cleavage required Mg2+ (optimally at 0.5–2.0 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.

ress: 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;

A, ethylenediaminetetraacetic acid; IDT, Integrated DNA Technologies; NSERC,esearch Council.

lsevier Ltd. All rights reserved.

trol

A A 1F 0N 6A 3N 9S8A(a)

961Endoribonuclease Active Site of Human APE1

Introduction

Con

WT

N68 D70 Y17 D21 F26 D28 H30D30

1 2 3 4 5 6 7 8 9 10

(b)

5´ 3´

AP-dsDNA

G

C

G

CG

C

A

GC

A

T

G

C

A

T

G

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A

TGC

AT

F

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T

G

C

A

TG

CA

TG

3´ 5´

18nt

5´

C

G

C

TG

CA

TG

9ntProduct

18 nt

9 nt

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 2–10) were incubated with5′-γ-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).

There is increasing evidence that endonucleolyticcleavage of mRNA plays a critical role in eukaryoticand mammalian gene expression.1–4 Recent reportson several enzymes possessing endoribonucleolyticactivities have highlighted their roles in mediatingthe posttranscriptional regulation of geneexpression.5–9 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 (2–5A) 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 β-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.19–23 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-κB,p53, c-Jun, and HIF).16,24 APE1 has been found topossess multiple DNA nuclease functions, including3′ phosphodiesterase,25 3′–5′ exonuclease,26 and 3′phosphatase 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,including the recently discovered single-stranded15

and abasic single-stranded31 RNA-cleaving activitiesof APE1.We have previously shown that H309N and E96A

mutants of APE1 have no RNA-cleaving activity,suggesting that the domain containing this enzy-

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 thestructural basis of RNA cleavage by APE1, as wellas for dissecting the significance of the endoribonu-clease function of APE1 in vivo.

962 Endoribonuclease Active Site of Human APE1

Results

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-dinic (AP) site cleavage activity of these APE1structural mutants was first assessed using apreviously employed 18 -bp AP-dsDNA substrate34

(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)19–23 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

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

Table 1. Summary of fold reduction in the endoribonucleasmutants as compared to WT APE1

APE1 structuralmutants

Fold reductionin AP-dsDNA incisiona

Foin AP

N68A 60023

D70A UDd

E96A 600–100,00021,30

Y171F 500021

D210N 25,00021

N212A UC19

F266A 620

D283N 1022

D308A 5–2521,33

H309N 100,00030

H309S UD

AB, abolished; UD, undetermined; and UC, unchanged activity as coa 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.23

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 tobind c-myc CRD RNA by electrophoretic mobilityshift assay (EMSA).Experiments were performed initially to optimize

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 RNA–APE1 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 RNA–APE1 complex (lanes 4–6; 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

e and AP-dsDNA incision activities of APE1 structural

ld reduction-dsDNA incisionb

Fold reduction in endoribonucleaseactivity against c-myc CRDc

AB AB6.7 ABUD AB15

AB ABAB ABUD UD30.4 ABAB UC1.4 ABUD AB15

AB UD

mpared to that of WT APE1.

1757UA1751UA1747UA

1742CA

1730UG1727CA

1775CA1773UA

1768CA1771CA

WT

N68A

D70A

Y171F

D210N

F266A

D283N

H309S

D308A

Control

1 2 3 4 5 6 7 8 9 10

(b)

c-myc 1705-1792 CRD RNA

(a)

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A C C A G A U C C U C C A A G C

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C

U

G

U

A U

A

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A

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G CC

A

A U

U

1775CA

1727CA

1730UG

5´ 3´

Fig. 2. Structural mutants of APE1 display reduced endoribonuclease activity. (a) The secondary structure andsequence of c-myc 1705–1792 CRD are shown. Arrows indicate the cleavage sites of WTAPE1. (b) Endoribonuclease assaywas carried out on c-myc CRD RNA with APE1 and its structural mutants, as described in Materials and Methods.Recombinant proteins (1.4 μM; lanes 2–10) were tested against 25 nM 5′-γ-32P-radiolabeled c-myc 1705–1792 CRD in atotal reaction volume of 20 μl for 25 min at 37 °C. Numbers on the right indicate the cleavage sites generated by APE1.

963Endoribonuclease Active Site of Human APE1

CRD RNA. Our findings on the progressive shift ofthe APE1–RNA 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 of0.87–125 nM,20,36–38 suggesting that APE1 has alower affinity for RNA substrates.We next evaluated the relative ability of the panel

of APE1 structural mutants to bind c-mycCRDRNA.We employed protein concentrations thatwere in thelinear range for binding to the RNA substrate byWTAPE1 (0–1412 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 2–4) and E96A(lanes 6–8) proteins exhibit similar binding affinitiesfor c-myc CRD RNA, with 50–60% of c-myc RNAbound. In contrast, H309N (lanes 10–12) showedreduced binding, with 20–35% of c-myc RNA bound.

We found that N68A (lanes 18–20), Y171F (lanes 22–24), and D210N (lanes 26–28) all displayed RNAbinding affinities comparable toWTAPE1 (lanes 2–4and 14–16). Interestingly, at lower concentrations(353 and 706 nM; lanes 30 and 31), the D283Nmutant protein showed a modest decrease inbinding, with 30–40% of c-myc RNA bound. At ahigher concentration (1412 nM; lane 32), the RNAbinding affinity of D283N became comparable tothat of WT APE1 (lanes 2–4 and 14–16; Fig. 3c).

APE1 D283N active-site mutation abolishesabasic single-stranded DNA cleavage activitybut retains abasic single-stranded RNAcleavage activity

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 the D283N mutant retainsstrong endoribonuclease activity on c-myc CRDRNA, comparable to that of the WT protein (Fig.2b). We therefore wanted to assess the effect of theD283N mutation on abasic single-stranded DNA(AP-ssDNA) and abasic single-stranded RNA (AP-ssRNA) cleavage, as WT APE1 possesses AP site

(a)

0 141242

3556

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WT E96A

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614

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1

0 1000 2000 3000 4000 5000 6000 7000 8000

[APE1] (nM)

% b

ou

nd

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100)

Fig. 3. Ability of APE1 and structural mutants to bind c-myc CRD RNA. Increasing amounts of WT APE1 or APE1structural mutants were incubated with 50 nM 32P internally radiolabeled c-myc 1705–1886 CRD RNA and examinedusing a total binding volume of 20 μl for 15 min at 35 °C in standard EMSA, as described in Materials and Methods. (a)EMSA shows the binding of WT APE1 from 0 to 7058 nM. (b) Binding activity of WT APE1 was quantified by comparingto the relative amount of radiolabeled unbound RNA shifted to the slower-migrating bound RNA. Data obtained werethen used to plot the saturation binding curve, as shown. (c) EMSA shows the ability of various concentrations of WTAPE1 (lanes 2–4 and 14–16), E96A (lanes 6–8), H309N (lanes 10–12), N68A (lanes 18–20), Y171F (lanes 22–24), D210N(lanes 26–28), and D283N (lanes 30–32) to bind 32P-labeled c-myc CRD RNA.

964 Endoribonuclease Active Site of Human APE1

incision activity on both forms of nucleic acid.31

Using single-stranded 34-mer substrates ofmatchingbase sequences but varying sugar compositions(deoxyribose versus ribose) (Fig. 4a), we comparedthe AP site incision activities of WT and D283NAPE1. We found that the single-stranded DNA(ssDNA) AP site incision activity of D283N wasseverely diminished (∼50-fold; WT-specific activityof 747+35×105 pmol min−1 mg−1 and D283N-specific activity of 15.5+11×105 pmol min−1 mg−1)(Fig. 4b and d), while the ssRNA AP site incisionactivity of D283N was only mildly affected (∼1.2-fold; WT-specific activity of 37+8×104 pmol min−1

mg− 1 and D283N-specific activity of 29 +7×104 pmol min−1 mg−1) (Fig. 4c and d). Theseresults are consistent with the differential incisionactivities observed for D283N on the AP sitecontaining dsDNA (Fig. 1) versus c-myc RNA (Fig. 2).

RNA catalysis mechanism of APE1

To determine whether WT APE1 generates RNAproducts with a 3′-PO4

2− or a 3′-OH group, weadopted the method described by Chernokalskayaet al. using snake venom exonuclease to digest onlyproducts bearing a 3′-OH.41 Figure 5a shows thatthe distinct product generated by RNase T1 (asteriskin lane 2) remained visible after digestion with snakevenom exonuclease (lane 3), consistent with the factthat RNase T1 generates products with 3′-PO4

2−. Incontrast, S1-nuclease-generated products (asterisksin lane 5) were completely degraded upon treatmentwith snake venom exonuclease (lane 6), consistentwith the notion that S1 nuclease generates productswith 3′-OH. The distinct decay products generatedby APE1 (asterisks in lane 9) were still visible upontreatment with snake venom exonuclease (lane 8),

Fig. 4. AP-ssDNA and AP-ssRNA endonuclease activities ofWT APE1 and D283N mutant. (a)Predicted secondary structures of34FDNA (left) and 34FRNA (right),as determined using Mfold. F de-notes the position of the abasic-siteanalog tetrahydrofuran. (b) AP-ssDNA endonuclease activities ofWT and D283N APE1. Time-coursereactions contained 15 pM APE1WT or D283N protein and 200 fmolof 5′-γ-32P-radiolabeled 34FDNAand were incubated at 37 °C forvarious times, as shown. S denotessubstrate position, and P denotesproduct position. (c) AP-ssRNAendonuclease activities of WT andD283N APE1. Time-course reac-tions contained 1.5 nM APE1 WTor D283N protein and 200 fmol of5′-γ-32P-radiolabeled 34FRNA andwere incubated at 37 °C for varioustimes, as shown. S denotes sub-strate position, and P denotes prod-uct position. (d) Graph depictingWT and D283N APE1 time-courseincision kinetics on 34-mer AP-ssDNA and 34-mer AP-ssRNA.Average values with standard de-viations from at least three inde-pendent experiments were plotted.

965Endoribonuclease Active Site of Human APE1

consistent with WT APE1 generating RNA productswith 3′-PO4

2− groups.In a general acid/base-mediated reaction mecha-

nism, which produces RNA intermediates with 3′-PO4

2−, a 2′-OH group is required as a potentialprovider of the catalytic nucleophile. To betterunderstand the mechanism of APE1 RNA catalysis,we implemented the use of two different RNAsubstrates: dOligoIA and OligoIA (Fig. 5c). Ourresults showed that when challenged with dOligoIA,containing a target uridine at base position 10 linkedto a deoxyribose moiety (dUpA), WT APE1 wasunable to produce any decay products (lanes 1–6; Fig.

5b). However, when challenged with OligoIA, con-taining a uridine at base position 10 linked to a ribosegroup (rUpA), and thus a 2′-OH, WT APE1 was ableto generate increasing amounts of the expectedcleavage product over time (lanes 7–12; Fig. 5b).Our results indicate that a 2′-OH on the sugar moietyis essential for APE1-mediated RNA catalysis.To further understand the mechanism of the

endoribonuclease activity of APE1 and to elucidatethe effect of the D283 mutation on APE1 RNAnuclease activity, we conducted the followingexperiment. We challenged the D283N APE1 mu-tant with dOligoIA and OligoIA, as described in Fig.

1 2 3 4 5 6 7 8 9 10 11 12

dOligo IA Oligo IA

rU

C

C

C

A

A

G

G

A

G

A

T

T

T

G

T T

5´ 3´

Oligo IA

dU

C

C

C

A

A

G

G

A

G

A

T

T

T

G

T T

5´ 3´

dOligo IA

min0 2.5 5 7.5 10 20 0 2.5 5 7.5 10 20

1 2 3 4 5 6 7 8 9

- - + - - + - + - 3’-5’ ExonucleaseAPE1

- S1 Nuclease- + +

+ ++ +

- - -- - -

- - - - - - -

- - -- - -

RNase T1

* *

***

*

*

(a)

(b)

(c) (d)

1 2 3 4 5 6

dOligo IA Oligo IA

min0 10 30 0 5 30

D283N

Fig. 5. RNA catalytic reactionmechanism of APE1. (a) APE1generates RNA products with 3′-PO4

2−. 5′-Labeled c-myc CRD RNA(350 fmol) was digested with 1 U ofRNase T1 (lanes 2 and 3) for 1 minat 37 °C or with 10 U of S1 nuclease(lanes 5 and 6) or 8.5 μM WT APE1(lanes 8 and 9) for 5 min at 37 °C onstandard endonuclease assay, asdescr ibed in Mater ia ls andMethods. The products were thenincubated with 3×10−5 U of snakevenom exonuclease for 10 min at25 °C (lanes 3, 6, and 8) andelectrophoresed on an 8% poly-acrylamide–urea gel. Asterisks des-ignate those products generated byRNase T1 (lane 2), S1 nuclease (lane5), or APE1 (lane 9) that weremonitored for exonuclease degra-dation. (b) Endoribonuclease activ-ity of APE1 requiring 2′-OH on thesugar moiety. APE1 (1.4 μM) wastested against 25 nM 5′-γ-32P-radi-olabeled dOligoIA (lanes 1–6) orOligoIA (lanes 7–12) in a totalreaction volume of 20 μl for varyingtime points at 37 °C. (c) Secondarystructures of dOligoIA and Oli-goIA, as predicted using Mfold.(d) D283N APE1 (1.4 μM) testedagainst 25 nM 5′-γ-32P-radiolabeleddOligoIA (lanes 1–3) or OligoIA(lanes 4–6) in a total reaction vol-ume of 20 μl for varying time pointsat 37 °C. The result shown isrepresentative of three independentexperiments.

966 Endoribonuclease Active Site of Human APE1

5. Our results showed that when D283N wasincubated with dOligoIA, no distinct product wasobserved (lanes 1–3; Fig. 5d). However, whenD283N APE1 was challenged with OligoIA, increas-ing amounts of the expected cleavage product wereobserved over time (lanes 4–6; Fig. 5d). These resultsare similar to what was observed with the WT APE1protein (Fig. 5b) and suggest that mutation of D283does not affect the ability of APE1 to utilize the 2′-OH in the sugar moiety for RNA catalysis.

Mg2+ modulates APE1 nuclease activities

To understand the role that Mg2+ plays in theendoribonuclease activity of WT APE1, we firstexamined the effect of increasing concentrations ofthe divalent metal on the APE1 cleavage of c-mycCRD RNA. APE1 exhibited RNA incision activity inthe absence of Mg2+ (lane 2; Fig. 6a) whiledisplaying the highest activity at 5 mM Mg2+ (lane4; Fig. 6a), consistent with our previous fluorometric

assay measuring the endoribonuclease activity ofAPE1.32 At concentrations greater than 5 mM (lanes5–7; Fig. 6a), Mg2+ caused a gradual decrease in theendoribonuclease activity of APE1.Mg2+ completelyinhibited the activity at 100 and 200 mM (lanes8 and 9; Fig. 6a). Interestingly, we also observed achange in the RNA cleavage pattern at 20 and50 mM Mg2+ (lanes 6 and 7; Fig. 6a), suggestingthat the metal ion may alter either the RNAsecondary structure or RNA binding by APE1. Toexplore the latter hypothesis, we employed EMSAto assess the role that Mg2+ plays in RNA complexformation by APE1. Figure 6b shows that neitherthe nature of the complex nor the binding affinityof APE1 was dramatically altered by high Mg2+

levels. We therefore conclude that the altered RNAcleavage pattern observed is likely due to alter-ations in the RNA secondary structure imposed byhigher concentrations of Mg2+.We next examined the effects of differential Mg2+

concentrations on the ssDNA and ssRNA AP site

1 2 3 4 5 6 7 8 9 10 11 12 13 14

- + - + - + - + - + - + - + APE1Mg (mM)2+0 2 5 10 20 50 100

(b)(a)2 5 10 20 50 10

020

00

1 2 3 4 5 6 7 8 9

0- + + + + + + + + APE1

Mg (mM)2+

(c) (d)

Fig. 6. Effects of magnesium on the endoribonuclease, RNA binding, AP-ssDNA, and AP-ssRNA endonucleaseactivities of APE1. (a) APE1 (1.4 μM; lanes 2–9) tested against 25 nM 5′-γ-32P-radiolabeled c-myc CRD RNA in a totalreaction volume of 20 μl for 25 min at 37 °C. Each lane contains a different magnesium concentration, as indicated. (b) WTAPE1 tested against 50 nM 32P internally radiolabeled c-myc CRD RNA in a total binding volume of 20 μl for 15 min at35 °C using standard EMSA at increasing concentrations of Mg2+. (c) Graph of relative AP-ssDNA incision byWTAPE1 atvarious Mg2+ concentrations. WT APE1 (15 pM) was incubated with 200 fmol of 5′-γ-32P-radiolabeled 34FDNA in abuffer containing 0 , 0.5 , 1 , 2 , 5 , 10 , or 20 mMMg2+ at 37 °C for 5 min. Average values with standard deviations from atleast three independent experiments are plotted relative to 1 mM Mg2+. (d) Graph of relative AP-ssRNA incision by WTAPE1 at various Mg2+ concentrations. WT APE1 (1.5 nM) was incubated with 200 fmol of 5′-γ-32P-radiolabeled 34FRNAin a buffer containing the designated Mg2+ concentration at 37 °C for 5 min. Average values with standard deviationsfrom at least three independent experiments are plotted relative to 1 mM Mg2+.

967Endoribonuclease Active Site of Human APE1

incision activities of WT APE1. Consistent withprevious results for dsDNA AP site endonucleaseactivity,33 APE1 required Mg2+ to cleave at an APsite in ssDNA. Optimal ssDNA AP endonucleaseactivity was observed at a concentration of 1 mMMg2+, with high levels of incision activity present atboth 0.5 and 2 mM Mg2+. A marked decrease inssDNA AP endonuclease activity occurred at Mg2+

concentrations of 5 and 10 mM, while 20 mM Mg2+

completely inhibited APE1 ssDNA AP site cleavage(Fig. 6c). When we examined ssRNAAP site incisionby APE1, we found that differential Mg2+ concen-trations affected cleavage much less drastically.Similar to the endoribonuclease activity (Fig. 6a),the ssRNA AP site cleavage activity of APE1 wasactive in the absence of Mg2+; indeed, the highestlevels of ssRNA AP site incision activity wereobserved at 0 mM Mg2+ (Fig. 6d). High levels ofssRNA AP site cleavage occurred at all tested Mg2+

concentrations (0.5–20 mM; Fig. 6d), suggesting that

the catalytic mechanism of APE1 RNA cleavagediffers from that used for AP-DNA cleavage anddoes not require Mg2+.

Discussion

We previously showed that amino acids H309 andE96, critical for the other known nuclease activities ofAPE1,29,33,42–44 are also important for the endoribo-nuclease activity of APE1 on c-myc CRD RNA.15 Thisobservation suggested that the RNA-cleaving activityofAPE1 residedwithin the same active site as its othernuclease activities. Furthermore, we reported that theRNA-cleaving activity of APE1 was active in theabsence of magnesium,32 in stark contrast to theabasic DNA endonuclease activity of APE1where themetal ion is absolutely essential. Such observationsprompted us to further investigate possible differ-ences in the mechanisms of RNA cleavage and the

968 Endoribonuclease Active Site of Human APE1

AP-DNA endonuclease activities of APE1. Moreover,we sought to identify unique RNA incision propertiesof APE1, which will ultimately allow for studies ofthis activity in cells. Our results herein reveal thatmost amino acid residues that are critical forAP-dsDNA incision are also important for theendoribonuclease activity of APE1. We found, how-ever, the following notable distinctions between theendoribonuclease/AP-RNA endonuclease activitiesand the AP-DNA endonuclease activities of APE1: (i)in contrast to its prominent role in dsDNA or ssDNAAP site endonuclease activity, D283 is not obviouslyinvolved in the RNA-cleaving activity of APE1, nor isit required for AP-ssRNA incision; (ii) unlike what isseen with AP-DNA substrates, Mg2+ is not requiredfor the endoribonuclease or AP-ssRNA endonucleaseactivities of APE1; and (iii) the RNA-cleaving activityof APE1 requires a 2′-OH on the sugar moiety of thenucleotide 5′ to the phosphodiester cleavage site. Thislatter finding is consistent with the observation thatAPE1 can endonucleolytically cleave RNA, leavingbehind a 3′-PO4

2− group.Employing well-established methods for asses-

sing various APE1 activities, we examined thecontributions of specific amino acid residues tosingle-stranded and double-stranded AP-DNAincision,34,42 RNA cleavage,15 and AP-ssRNAincision.31 The comparable degree of reduction inactivity presumably corresponds to the importanceof a given residue in the various nuclease activitiesof APE1 and suggests the presence of a commonactive site (Table 1). The most severe reductions inRNA incision activity were observed with N68A,Y171F, D210N, and H309S mutations (Table 1).Although there are multiple proposed reaction

mechanisms, N68 and D210 have been found to playcritical roles in catalyzing AP-DNA incision, possi-bly with N68 hydrogen bonding to D210 and H309to establish the optimal active-site environment andwith D210 participating in the generation of anucleophile.21,23 D70A and D308A displayed mod-erate decreases in AP-dsDNA activity (Fig. 1a) but acomplete abrogation of RNA incision activity (Fig.2b). These two residues have been predicted tocoordinate metal ions21 during abasic DNA incisionand are thought to orient the substrate, to stabilizethe transition state, and to facilitate the dissociationof the product. Overall, the results indicate thatAPE1 shares active-site residues participating in thecleavage of AP-DNA, RNA, and AP-RNA. Oneparticular residue for which this statement does nothold true is D283, where AP sites containing dsDNA(Fig. 1a) and ssDNA (Fig. 4a) activities wereabolished by mutation to asparagine, while endor-ibonuclease (Fig. 2b) and AP-ssRNA incision (Fig.4b) activities remained robust. In view of the role ofD283 in the maintenance of the D283-H309 dyad,22

our results indicate that this residue coordination isnot critical for RNA incision but is vital for efficient

AP-DNA incision (Figs. 1a and 4b).22 Our findingthat the D283N APE1 is still capable of cleavingOligoIA substrate (Fig. 5d) provides further supportfor this notion.Previously, H309N was shown to have a 2-fold

reduction in its affinity for AP-dsDNA.39 This is inline with our results, which showed diminishedRNA binding ability compared to WT APE1 (Fig.3c). Such a result indicates that H309 is involved inthe RNA binding process and that mutation at thisposition leads to reduced complex formation andsubsequent reduction in incision activity. Althoughour results demonstrated that residues N68, E96A,Y171, and D210 (Fig. 2) are critical for RNA incisionactivity, mutation at these residues had no effect onRNA binding (Fig. 3), suggesting that these residuesdo not affect the RNA binding step. This conclusionis consistent with their predicted importance in thecatalytic reaction mechanism of RNA incision.In addition to H309, D283N mutation can affect

RNA binding. At low concentrations (lanes 18 and19; Fig. 3c), D283N appears to display reduced RNAbinding. This observation appears to be consistentwith a previous report that showed a reduced abilityof the mutant to bind AP-dsDNA at lower proteinconcentrations.39 However, D283 does not appear tobe a major determinant in RNA or AP-ssRNAcleavage, as the D283N mutation did not drasticallyaffect either RNA incision activity of APE1.In summary, our results showed that, apart from

D283, amino acid residues that are critical forAP-DNA incision are also important for the endor-ibonuclease activity of APE1. These residues mostlikely play key roles in the catalytic reaction steps ofRNA incision because mutation at these positionsdid not significantly affect RNA binding. Ourfindings further confirm that the endoribonucleaseactivity of APE1 shares a common active site withthe other nuclease activities; however, our resultsalso show that the mechanisms for incising RNA,AP-ssRNA, AP-dsDNA, or AP-ssDNA are notentirely identical. Such distinctions, particularlythe observations with the D283 mutation, couldwell be sufficient to provide opportunities forunderstanding the contributions of the endoribonu-clease function of APE1 to controlling gene expres-sion in cells.

Materials and Methods

Purification of recombinant proteins

The recombinant APE1 structural mutants (N68A,D70A, Y171F, F266A, D283N, D308A, and H309S), aswell as the WT APE1 protein used for comparativestudies, were overexpressed in bacteria and purified asdescribed previously.21 Briefly, following ion-exchangechromatography,21 APE1 protein fractions were pooled,

969Endoribonuclease Active Site of Human APE1

concentrated by 80% (wt/vol) ammonium sulfate, andfractionated on a Bio-Rad BioSil SEC 125-5 gel-filtrationcolumn (7.8 mm×300 mm) in 50 mM Na-Hepes (pH 7.5),5% glycerol (wt/vol), 0.1 mM ethylenediaminetetraaceticacid (EDTA), and 0.1 mM DTT. The flow rate was0.25 ml/min. Proteins were detected by ultraviolet absor-bance at 280 nm. APE1 proteins were dialyzed overnightagainst 50mMTris–HCl (pH7.9), 50mMKCl, 20%glycerol,1 mM PMSF, and 0.1 mM DTT and stored at −70 °C.

Preparation of radiolabeled nucleic acids

The plasmids pGEM4Z-myc 1705–1792 and pGEM4Z-myc 1705–1886 were linearized and transcribed in vitro, asdescribed previously, to synthesize human c-myc CRDRNA corresponding to nucleotides 1705–1792 and 1705–1886. Oligonucleotides OligoIA (5′-CAAGGTAG-TrUATCCTTG-3′) and dOligoIA (5′-CAAGGTAGT-dUATCCTTG-3 ′ ) corresponded to c-myc CRDnucleotides 1742–1757 andwere synthesized by IntegratedDNA Technologies (IDT) Inc. (Coralville, IA). rU and dUindicate ribose and deoxyribose moieties linked to theuridine bases, respectively. The RNAs were 5′-end radi-olabeled with [γ-32P]ATP using T4 polynucleotide kinase.For internal labeling, [α-32P]UTP (Perkin-Elmer, Boston,MA) was used during transcription.15 Tetrahydrofuran (F;an abasic-site analog)-containing ssDNA and ssRNAoligonucleotides were purchased from IDT Inc. andradiolabeled at the 5′ end using [γ-32P]ATP and T4polynucleotide kinase (New England Biolaboratories,Ipswich, MA). Unincorporated [γ-32P]ATP was removedby centrifugation through a G-50 spin column (GEHealthcare, Quebec). Labeled oligonucleotides wererun on a 12% native polyacrylamide gel, and thenintact full-length bands were excised and eluted into25 mM 4-morpholinepropanesulfonic acid (Mops;pH 7.2) by incubation at 4 °C overnight.

In vitro assay for AP-dsDNA incision

The established protocol for APE1 AP-dsDNA endo-nuclease assay was used with minor modifications.34 The18-mer oligonucleotide 5′-GTCACCGTGFTACGACTC-3′that contains the model analog of an abasic site,tetrahydrofuran (F), was used. This oligonucleotide andits complementary anti-sense strand 5′-GAGTCGTAA-CACGGTGAC-3′ were synthesized by IDT Inc. Theoligonucleotide containing the abasic site was 5′-endradiolabeled with [γ-32P]ATP using T4 polynucleotidekinase. The reaction was stopped by heating at 95 °C for2 min, followed by hybridization to a 7× molar excess ofthe anti-sense strand at room temperature for 60 min andthen at 4 °C overnight. The AP-dsDNA incision assaycontains 15 μl of reaction mixture consisting of80,000 cpm, equivalent to 0.1 pmol of abasic DNA,25 pg (0.7 fmol) of APE1, 50 mM Tris–HCl (pH 8),50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 2 mM MgCl2,and 100 μg/ml bovine serum albumin. The reaction wascarried out at 37 °C for 3 min. Thirty microliters ofloading dye (9 M urea, 0.2% xylene cyanol, and 0.2%bromophenol blue) was added to the reaction samplesand then subjected to electrophoresis on an 8% poly-acrylamide–7 M urea gel.

AP site incision assay for ssDNA and ssRNA

Recombinant WT and D283N APE1 were incubatedwith PAGE-purified 5′-32P-labeled abasic single-stranded34FDNA (5 ′ -CTGCAGCTGATGCGCFGTACG-GATCCCCGGGTAC-3′) or 34FRNA (5′-CUGCAGCU-GAUGCGCFGUCCGGAUCCCCGGGUAC-3′) at 37 °Cin 25 mM Mops (pH 7.2), 100 mM KCl, and 1 mM MgCl2for the specified times (0, 2.5, 5, 10, and 20 min). Forreactions examining differential Mg2+ concentration onincision activity, buffers contained 25 mM Mops (pH 7.2);100 mM KCl; and 0 , 0.5 , 1 , 2 , 5 , 10 , or 20 mM MgCl2,respectively. These reactions were incubated at 37 °C for5 min. Reactions with 34FDNA contained 15 pM APE1,and reactions with 34FRNA contained 1.5 nM APE1.Reactions were inhibited by the addition of stop buffer(95% formamide, 20 mM EDTA, 0.5% bromophenol blue,and 0.5% xylene cyanol) and then heated at 95 °C for5 min. Reaction products were resolved by a 15%polyacrylamide urea denaturing gel, imaged, and quan-tified using a Typhoon PhosphorImager and ImageQuantTL software (GE Healthcare, Piscataway, NJ). For34FRNA, please note that about 20% background ispresent in all lanes from incubation with the reactionmixture and was subtracted from the total productformation at all time points for bothWT and D283NAPE1.

In vitro endoribonuclease assay

The standard 20-μl reaction mixture used for this assayincluded 2 mM DTT, 1.0 U of RNasin, 2 mM magnesiumacetate, 10 mM Tris–HCl (pH 7.4), and 25 nM 5′-32P-radiolabeled RNA (∼5×104 cpm). Reactions were incu-bated for 25 min at 37 °C, unless otherwise indicated.Forty microliters of loading dye (9 M urea, 0.2% xylenecyanol, and 0.2% bromophenol blue) was added to thereaction samples, and then 10 μl of the reaction mixturewas subjected to electrophoresis in an 8% polyacryl-amide–7 M urea gel. Gels were then dried and subjectedto PhosphorImaging using a Cyclone PhosphorImager(Perkin-Elmer, Woodbridge, ON).

Electrophoretic mobility shift assays

EMSA binding buffer [5 mM Tris–Cl (pH 7.4), 2.5 mMEDTA (pH 8.0), 2 mM DTT, 5% glycerol, 0.1 mg/mlbovine serum albumin, 0.5 mg/ml yeast tRNA, and 5 U ofRNasin] was prepared on ice prior to each experiment. Inorder to facilitate RNA denaturation and renaturation, weheated 30 nM internally labeled [32P]c-myc CRD RNAnucleotides 1705–1886 to 50 °C for 5 min and cooled themto room temperature before adding them to the EMSAbinding buffer. The EMSA binding buffer containingradiolabeled RNA was then incubated with purifiedrecombinant protein in a 20-μl reaction volume at 35 °Cfor 15 min. Two microliters of EMSA loading dye[250 mM Tris–Cl (pH 7.4), 0.2% bromophenol blue, and0.2% xylene cyanol] was added to each reaction, and 10 μlof the EMSA reaction was loaded onto an 8% nativepolyacrylamide gel and resolved at 25 mA for 2 h. Gelswere then subjected to PhosphorImaging. The EMSAsaturation binding experiments were carried out asdescribed above, and the dissociation constant Kd for

970 Endoribonuclease Active Site of Human APE1

APE1 against RNA was determined by fits to the Hillequation [Eq. (1)], where Kd is the dissociation constant,[L] is the concentration of APE1 (in nanomolars), and n isthe Hill coefficient:

BoundTotal

=1Kd

� �L½ �n

1 + 1Kd

� �L½ �

� �n ð1Þ

All saturation binding data were analyzed by densi-tometry of autoradiographs using Optiquant software(Perkin-Elmer). For each reaction (an individual lane), thetotal activity was determined by summing the total countsin bound complexes with the total counts present in theunbound fraction. In the lane with no protein added (lane1; Fig. 1a), the total activity was determined by drawing asquare box around the unbound fraction. The total countsin unbound fractions in the lanes with proteins (lanes 2–6;Fig. 1a) were determined by drawing a similarly sizedsquare box around the similar mobility, as in lane 1 on thegel. This analysis allowed for the calculation of thepercentage of RNA bound to the protein. Data were fittedto Eq. (1) and plotted using KaleidaGraph (SynergySoftware, Reading, PA).

Acknowledgements

This work was supported by Discovery Grant227158 from the Natural Sciences and EngineeringResearch Council (NSERC) to C.H.L. W.-C.K. wasthe recipient of a Pacific Century Scholarship, aMichael Smith Foundation of Health ResearchJunior Graduate Scholarship Award, and anNSERC Canada Graduate Scholarship. C.U. wasthe recipient of an NSERC Undergraduate StudentResearch Award. This work was also supported bythe Intramural Research Program of the NationalInstitute on Aging, National Institutes of Health(D.M.W.).

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