Intragenic suppression of an active site mutation in the human apurinic/apyrimidinic endonuclease

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<ul><li><p>Intragenic Suppression of anthe Human Apurinic/Apyrimi</p><p>Tadahide Izumi1,2, Jedrzej Malecki2, Mh</p><p>Galveston, TX 77555-1079, USA</p><p>Edmonton, Canada T6G 1Z2</p><p>mim</p><p>n2,tan</p><p>Escherichia coli, led to isolation of an intragenic suppressor with a secondsite mutation, K98R. Although the K of the suppressor mutant was</p><p>E96A mutation affects only the DNA-binding step, but not the catalytic</p><p>(eoat</p><p>base excision repair in DNA (BER), are genotoxic Cunningham, 1990). APEs also possess a 30 DNA</p><p>Article No. jmbi1999.2573 available online at on J. Mol. Biol. (1999) 287, 4757and mutagenic (Wallace, 1994). AP endonucleases(APE) incise DNA strands 50 to the AP sites and</p><p>phosphoesterase activity and thus remove abnor-mal 30 termini such as 30-phosphoglycolate (30-PG)which are generated by ROS at DNA strand breaksand prevent DNA repair synthesis (Doetsch &amp;Cunningham, 1990). Escherichia coli has two APEgenes, exonuclease III (Xth) and endonuclease IV(Nxfo), encoded by xth and nfo, respectively. Thexth nfo double mutant is highly sensitive to such</p><p>Abbreviations used: AP site, apurinic/apyrimidinicsite; BER, DNA base excision repair; DTT, dithiothreitol;EDTA, ethylenediaminetetra-acetic acid; hAPE-1, humanapurinic/apyrimidinic endonuclease 1; MMS, methylmethanesulfonate; nfo, endonuclease IV; PBS, phosphate*Corresponding author</p><p>Introduction</p><p>Apurinic and apyrimidinicgenerated either by spontandepurination or as intermediE-mail address of the</p><p>buffered saline; 30-PG, 30-phosphoglexonuclease III; APE, apyrimidinic</p><p>0022-2836/99/11004711 $30.00/0step of the enzyme. The 30 DNA phosphoesterase activities of the wild-type and the suppressor mutant were also comparable. No global changeof the protein conformation is induced by the single or double mutations,but a local perturbation in the structural environment of tryptophan resi-dues may be induced by the K98R mutation. The wild-type and suppres-sor mutant proteins have similar Mg2 requirement for activity. Theseresults suggest a minor perturbation in conformation of the suppressormutant enabling an unidentified Asp or Glu residue to substitute forGlu96 in positioning Mg2 during catalysis. The possibility that Asp70 issuch a residue, based on its observed proximity to the metal-binding sitein the wild-type protein, was excluded by site-specific mutation studies.It thus appears that another acidic residue coordinates with Mg2 in themutant protein. These results suggest a rather flexible conformation ofthe region surrounding the metal binding site in hAPE-1 which is notobvious from the X-ray crystallographic structure.</p><p># 1999 Academic Press</p><p>Keywords: DNA repair; AP endonuclease; 30phosphoesterase; missensemutation; site-directed mutagenesis</p><p>AP) sites in DNA,us and oxidative</p><p>e products during</p><p>produce 30 OH termini (Wallace, 1994; Doetsch &amp;Cunningham, 1990). Such enzymes are distin-guished from AP lyases which were originallyclassified as type I and generate 30 phospho a,bunsaturated aldehyde via b-elimination (Doetsch &amp;3Experimental OncologyCross Cancer Institute</p><p>m</p><p>about sixfold higher than that of the wild-type enzyme, their kcat valueswere similar for AP endonuclease activity. These results suggest that theMichael Weinfeld3, Jeff H. Hill1, J. C</p><p>1Sealy Center for MolecularScience and 2Department ofHuman Biological Chemistryand Genetics, University ofTexas Medical Branch</p><p>The apurinic/apyriconserved sequencemotif, LQE96TK98 iin coordinating Mginactive E96A muing author:</p><p>ycolate; xth,exonuclease.Active Site Mutation indinic Endonuclease</p><p>. Ahmad Chaudhry3</p><p>ing Lee2 and Sankar Mitra1,2*</p><p>dinic endonucleases (APE) contain several highlyotifs. The glutamic acid residue in a consensus</p><p>human APE (hAPE-1), is crucial because of its rolean essential cofactor. Random mutagenesis of thet cDNA, followed by phenotypic screening ingenotoxic agents as methyl methanesulfonate(MMS) and reactive oxygen species which lead tothe production of AP sites and 30-blocked termini</p><p># 1999 Academic Press</p></li><li><p>(Doetsch &amp; Cunningham, 1990; Cunningham et al.,1986).</p><p>Only one APE gene, APE-1 (originally namedAPEX, APE, HAP1, and Ref-1), has been identifiedand cloned in mammalian cells; the protein is ahomolog of E. coli Xth (Demple et al., 1991; Robson&amp; Hickson; 1991; Seki et al., 1992; Xanthoudakiset al., 1992). This enzyme accounts for most of thecellular APE activity, and its activation in responseto oxidative stress has been recently reported(Fung et al., 1998; Ramana et al., 1998). Besides theDNA repair function, the human APE-1 (hAPE-1)possesses two other unrelated functions. The pro-tein was identified as a redox-enhancing factor,Ref-1, which reductively activates AP-1 and p53transcription factors (Xanthoudakis et al., 1992;Jayaraman et al., 1997). Indeed, the N terminusdomain has recently been reported to interact withthioredoxin which appears to be needed for redoxreactivation of the Ref-1 activity (Qin et al., 1996;Hirota et al., 1997). The hAPE-1 was also identifiedas a corepressor of the parathyroid hormone geneand of its own gene (Okazaki et al., 1994; Izumiet al., 1996). Although the biological significanceof the multiple, unrelated properties of hAPE-1is not yet clear, the fact that the gene is essential</p><p>dues, respectively) led to total loss of the APEactivity (Izumi &amp; Mitra, 1998).</p><p>Alignment of the amino acid sequences of themembers of the APE family shows several highlyconserved motifs (Seki et al., 1992). One of these isthe GXDHCP sequence in which the histidine resi-due was identified as the catalytic residue(Figure 1). Another motif, LQETK, is also con-served in most APE enzymes (Figure 1). Indeed,elucidation of the tertiary structure of hAPE-1 andXth by X-ray crystallography led to the predictionthat Glu96 (E96) in the motif LQE96TK98 in hAPE-1 is essential for endonuclease activity because ofits role in coordinating the cofactor Mg2 (Barzilayet al., 1995a,b; Gorman et al., 1997). Substitution ofE96 with alanine (E96A) caused loss of the endonu-clease activity. However, a small activity of E96Awas observed in the E96A mutant protein (about400 times less than that of the wild-type) in con-trast to other active site mutants, such as H309N,of which the activity was at least 2000-fold lessthan that of the wild-type protein (Barzilay et al.,1995a,b). Because the metal ion cofactor is absol-utely required for the reaction (Gorman et al.,1997), we considered it likely that other amino acidresidues were also involved in the binding of Mg2</p><p>48 Intragenic Suppression of Human APE-1for the early embryonic development in mice(Xanthoudakis et al., 1996) indicates its essentialfunctions in cells.</p><p>Distinct regions in the hAPE-1 polypeptide areresponsible for the endonuclease/30-phosphoester-ase and gene regulatory activities (Xanthoudakiset al., 1994). The 60 amino acid residues at the Nterminus, which are not conserved in Xth, areinvolved in AP-1 activation and dispensable forthe APE activity (Walker et al., 1993; Izumi &amp;Mitra, 1998). Further truncation of the protein fromboth N and C termini (80 and five amino acid resi-with six coordinate bonds, and thus may beresponsible for the residual activity of the E96Amutant. Participation of multiple residues in themetal binding was also reported in E. coli RNaseH (Kashiwagi et al., 1996; Uchiyama et al., 1994).We entertained the possibility that a second mis-sense mutation in E96A could induce a subtle con-formational change in the neighborhood of theactive site surface that would allow positioning ofother residues in order to coordinate Mg2 moreefficiently, and thereby restore the catalytic activityof the protein. Such a mutant protein could pro-</p><p>Figure 1. Homology of polypep-tide sequences of APEs. Only thehomology of residues 61 to 99 andthe C-terminal segment of hAPE-1are shown. Sequences in Swiss-plotdatabase were aligned with GCG9.0. Names on the left are the entrynames in Swiss-Plot. The numbersin the second column denote thestarting residue number. The APendonuclease activity in hAPE-1 islost after deletion of the shadedregions (Izumi &amp; Mitra, 1998). TheE96 and K98 residues of hAPE-1and the two highly conservedmotifs are shown in bold letters.</p></li><li><p>vide significant insight into the nature of metalbinding to the essential DNA repair enzyme inmammals. Here, we report isolation and character-ization of such a missense suppressor of the E96Amutant which restored the AP endonuclease and30-PG-removing activity. Our results support themodel of participation of multiple acidic residuesin coordinating Mg2 and thereby the presence ofa flexible conformation of the protein.</p><p>Results</p><p>Isolation of intragenic suppressor mutant ofE96A; phenotypic rescue of E. coli by hAPE-1</p><p>APE is involved in the repair of DNA damageinduced by MMS, because AP sites are producedas intermediates during repair process(Cunningham et al., 1986). The human APE-1 wasshown to be able to complement xth nfo-negativeE. coli (Demple et al., 1991; Robson &amp; Hickson,1991; Izumi &amp; Mitra, 1998). We have recentlyestablished a phenotypic rescue system in E. colithat provides a sensitive screening procedure foridentifying active versus inactive hAPE-1 (Izumi &amp;Mitra, 1998).</p><p>well as H309A missense mutants were inactive(Barzilay et al., 1995b). Then we utilized the pheno-typic rescue strategy to examine the possibility ofintragenic suppression of E96A. An expressionplasmid cDNA containing E96A was randomlymutagenized by PCR (Spee et al., 1993), and intro-duced into xth nfo E. coli by transformation.Resistant clones were isolated after challenging thebacteria with MMS treatment (Figure 2). Our pre-liminary experiments showed that the original pro-tocol by Spee et al. (1993) yielded high frequency ofmultiple mutations in control experiments (datanot shown). Because multiple mutations are unde-sirable for our purpose, we modified the protocolby omitting dITP and reducing the concentrationof one dNTP to 40 mM, while maintaining the con-centration of the other three dNTPs at 200 mM(Spee et al., 1993). Four independent PCR reactionswith limiting concentration of different dNTPswere carried out so that misincorporation couldoccur at all sites. Of the approximately 2 104independent clones screened, 16 clones wereselected on the basis of MMS resistance of the hostbacteria. A high level of cellular protection wasobserved in the selected clones compared to thebacteria harboring the E96A mutant plasmid(Figure 3). The DNA sequence was determined forfour of them, and in all cases Lys98 was found to</p><p>Intragenic Suppression of Human APE-1 49Figure 2. (a) Site-directed mutagenesis of the hAPE-1by PCR. The 50 and 30 vector primers flanking thecDNA and two primers for each mutagenesis areshown. Newly introduced EcoRV (E96A) and NsiI(H309A) sites, as well as an endogenous site (BglII forD70N), are underlined. Silent mutations are shown inbold letters. After amplification by PCR, the fragmentswere inserted into the NdeI-SalI site in pIZ42 (Izumi &amp;Mitra, 1998). (b) A diagram of the random mutagenesisprocedure. The same vector primers as in (a) were used.We examined APE activity of two mutant pro-teins, E96A and H309A, in the cell toxicity assay(Figures 2 and 3). Both cDNAs failed to provideresistance to the E. coli strain against MMS. Noactivity was detected in either crude extracts(Figure 4(b)). We thus concluded that the E96A as</p><p>Figure 3. Phenotypic rescue of E. coli BW528 withwild-type (W) and mutant APEs. Details of MMS treat-ment were given in Materials and Methods. Vec, theempty vector control.</p></li><li><p>the level of expression and solubility of the mutantprotein were indistinguishable from those of thewild-type (Figure 4(a)), suggesting that no signifi-cant change in expression of the protein or in itsglobal conformation was caused by the mutation;this was further confirmed by circular dichroismand intrinsic fluorescence studies of the wild-typeand mutant proteins as described below. AP endo-nuclease activity in the crude extract of E. coliexpressing the suppressor mutant protein wasfound to be about half that in bacteria expressingthe wild-type protein (Figure 4(b)). We have con-cluded therefore that the E96A K98R doublemutant regained the AP endonuclease activitydespite the loss of E96, the residue predicted to beessential for Mg2 coordination (Gorman et al.,1997; Barzilay et al., 1995b).</p><p>To examine if such intragenic suppression is acommon phenomenon among the APEs, the corre-sponding glutamic acid (E34) and lysine (K36) resi-dues in the LQETK motif of the E. coli Xth proteinwere changed to Ala and Arg, respectively, by site-specific mutagenesis and the mutants were tested</p><p>50 Intragenic Suppression of Human APE-1be replaced with an arginine residue (K98R, AAAto AGA), while the original missense mutationremained unchanged. When expressed in E. coli,</p><p>Figure 4. AP endonuclease activity of wild-type andmutant proteins of hAPE-1. (a) Immunoblot assay ofwild-type, E96A, and E96A K98R expressed in E. coliBW528. The same amount of crude extracts was ana-lyzed by SDS-PAGE (12 % acrylamide) and subsequentimmunoblot assay (Izumi &amp; Mitra, 1998). Total and sol-uble fractions were analyzed separately. (b) AP endonu-clease activity was measured in crude lysates using atetrahydrofuran-containing 43mer the cell protection assay. While the E34A mutantwas inactive in cell survival assay as expected(Figure 5(a)), the defect was again suppressed byintroduction of the secondary missense, K36R,although the effect was not as pronounced as thatobserved for hAPE-1.</p><p>Characterization of the mutant protein and theeffect of Mg2 on incision activity</p><p>To further characterize the suppressor mutant onhAPE-1, we purified the wild-type and the mutantproteins to near homogeneity (Figure 6(a)). TheE. coli xth nfo strain (BW528) was lysogenized withl-DE3, a lambda phage containing the T7 RNApolymerase gene inducible by IPTG. Thus, the pur-ified APE protein did not contain any background</p><p>Figure 5. Phenotypic rescue ofE. coli BW528 with wild-type (W)and mutant APEs. (a) E. coli BW528with wild-type xth gene, E34A, andE34A K36R in pIZ42 were treatedwith MMS. (b) hAPE-1 cDNA withmissense mutations were subjectedto phenotypic rescue assay.W, wild-type hAPE-1; Vec, controlempty vector.</p></li><li><p>Intragenic Suppression of Human APE-1 51activity of class II AP endonucleases from E. coli(Cunningham et al., 1986). The DNA strand-clea-vage assay was highly specific for the endonu-clease activity of the recombinant APEs because ofthe use of tetrahydrofuran as a substrate. This sub-strate, unlike an intact AP site, is resistant to clea-vage via b elimination by contaminating AP lyases(Takeshita et al., 1987). Barzilay et al. (1995b)showed that although the E96A mutant lostactivity in low concentration of Mg2 (0.1 mM), theactivity was restored when the Mg2 concentrationin the reaction was increased to 2 mM. We exam-ined the effect of Mg2 concentration on theactivity of the mutant protein. As shown inFigure 6(b), the relative activity of the suppressormutant protein...</p></li></ul>