Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch
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Abstract:
Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base. The biological outcome is the prevention of C/G→A/T transversions. The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood. In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex. After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity. The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA. It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh–DNA complex. This stimulation is independent of the catalytic activity of Ape1. Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency. Our study suggests that protein–protein interactions may occur in vivo to achieve efficient BER of A/GO.Keywords:
AP endonuclease
MUTYH
Base loss is common in cellular DNA, resulting from spontaneous degradation and enzymatic removal of damaged bases. Apurinic/apyrimidinic (AP) endonucleases recognize and cleave abasic (AP) sites during base excision repair (BER). APE1 (REF1, HAP1) is the predominant AP endonuclease in mammalian cells. Here we analyzed the influences of APE1 on the human BER pathway. Specifically, APE1 enhanced the enzymatic activity of both flap endonuclease1 (FEN1) and DNA ligase I. FEN1 was stimulated on all tested substrates, regardless of flap length. Interestingly, we have found that APE1 can also inhibit the activities of both enzymes on substrates with a tetrahydrofuran (THF) residue on the 5'-downstream primer of a nick, simulating a reduced abasic site. However once the THF residue was displaced at least a single nucleotide, stimulation of FEN1 activity by APE1 resumes. Stimulation of DNA ligase I required the traditional nicked substrate. Furthermore, APE1 was able to enhance overall product formation in reconstitution of BER steps involving FEN1 cleavage followed by ligation. Overall, APE1 both stimulated downstream components of BER and prevented a futile cleavage and ligation cycle, indicating a far-reaching role in BER.
AP endonuclease
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Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5′-endonuclease activity and 3′-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex+/–). Our data indicate that Apex+/– mice are indeed APE-haploinsufficient, as exhibited by a 40–50% reduction (p < 0.05) in APE mRNA, protein, and 5′-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase β and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase β-dependent BER response to cellular stress. Apurinic/apyrimidinic (AP) endonuclease (APE) is a multifunctional protein possessing both DNA repair and redox regulatory activities. In base excision repair (BER), APE is responsible for processing spontaneous, chemical, or monofunctional DNA glycosylase-initiated AP sites via its 5′-endonuclease activity and 3′-"end-trimming" activity when processing residues produced as a consequence of bifunctional DNA glycosylases. In this study, we have fully characterized a mammalian model of APE haploinsufficiency by using a mouse containing a heterozygous gene-targeted deletion of the APE gene (Apex+/–). Our data indicate that Apex+/– mice are indeed APE-haploinsufficient, as exhibited by a 40–50% reduction (p < 0.05) in APE mRNA, protein, and 5′-endonuclease activity in all tissues studied. Based on gene dosage, we expected to see a concomitant reduction in BER activity; however, by using an in vitro G:U mismatch BER assay, we observed tissue-specific alterations in monofunctional glycosylase-initiated BER activity, e.g. liver (35% decrease, p < 0.05), testes (55% increase, p < 0.05), and brain (no significant difference). The observed changes in BER activity correlated tightly with changes in DNA polymerase β and AP site DNA binding levels. We propose a mechanism of BER that may be influenced by the redox regulatory activity of APE, and we suggest that reduced APE may render a cell/tissue more susceptible to dysregulation of the polymerase β-dependent BER response to cellular stress. Apurinic/apyrimidinic (AP) 1The abbreviations used are: AP, apurinic/apyrimidinic; APE, apurinic/apyrimidinic endonuclease; BER, base excision repair; β-pol, polymerase β; MFG, monofunctional glycosylase; dRP, 5′-deoxyribose 5-phosphate; EMSAs, electrophoretic mobility shift assays; DTT, dithiothreitol; PBS, phosphate-buffered saline; BSA, bovine serum albumin; RT, reverse transcriptase; I.D.V., integrated density value; ROPS, random oligonucleotide primed synthesis; ASB, aldehyde-reactive probe slot blot. endonuclease (APE) is a multifunctional protein involved in the maintenance of genomic integrity and in the regulation of gene expression. After the initial discovery in Escherichia coli (1Verly W.G. Paquette Y. Can. J. Biochem. 1972; 50: 217-224Google Scholar), APE was purified from calf thymus DNA and extensively characterized as an endonuclease that cleaves the backbone of double-stranded DNA containing AP sites (2Ljungquist S. Lindahl T. J. Biol. Chem. 1974; 249: 1530-1535Google Scholar, 3Ljungquist S. Andersson A. Lindahl T. J. Biol. Chem. 1974; 249: 1536-1540Google Scholar). APE homologues were subsequently identified and characterized in many organisms, including yeast as APN1 (4Popoff S.C. Spira A.I. Johnson A.W. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4193-4197Google Scholar), mice as Apex (5Seki S. Akiyama K. Watanabe S. Hatsushika M. Ikeda S. Tsutsui K. J. Biol. Chem. 1991; 266: 20797-20802Google Scholar, 6Seki S. Ikeda S. Watanabe S. Hatsushika M. Tsutsui K. Akiyama K. Zhang B. Biochim. Biophys. Acta. 1991; 1079: 57-64Google Scholar), and humans as HAP1 (7Robson C.N. Hickson I.D. Nucleic Acids Res. 1991; 19: 5519-5523Google Scholar). In addition to its major 5′-endonuclease activity, APE also expresses minor 3′-phosphodiesterase, 3′-phosphatase, and 3′ → 5′-exonuclease activities (8Wilson III, D.M. Sofinowski T.M. McNeill D.R. Front. Biosci. 2003; 8: D963-D981Google Scholar), the biological significance of which is controversial (9Lebedeva N.A. Khodyreva S.N. Favre A. Lavrik O.I. Biochem. Biophys. Res. Commun. 2003; 300: 182-187Google Scholar). Independent of its discovery as a DNA repair protein, APE was also characterized as REF-1, for redox factor-1, a redox activator of cellular transcription factors (10Xanthoudakis S. Curran T. EMBO J. 1992; 11: 653-665Google Scholar, 11Xanthoudakis S. Miao G. Wang F. Pan Y.C. Curran T. EMBO J. 1992; 11: 3323-3335Google Scholar, 12Evans A.R. Limp-Foster M. Kelley M.R. Mutat. Res. 2000; 461: 83-108Google Scholar). Although the molecular detail of APE redox activity is still unclear (13Ordway J.M. Eberhart D. Curran T. Mol. Cell. Biol. 2003; 23: 4257-4266Google Scholar), the discovery of APE as a regulator of transcriptional activity may underscore the importance of its involvement in cellular stress-response pathways. APE is the primary enzyme responsible for recognition and incision of non-coding AP sites in DNA arising as a consequence of spontaneous, chemical, or DNA glycosylase-mediated hydrolysis of the N-glycosyl bond initiated by the base excision repair (BER) pathway. These lesions are particularly common, arising at the rate of ∼50,000–200,000 AP sites per cell per day under normal physiological conditions (14Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225Google Scholar, 15Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526Google Scholar). Unrepaired AP sites may threaten genomic stability by serving as blocks to DNA replication (16Schaaper R.M. Kunkel T.A. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 487-491Google Scholar), by stalling RNA polymerase II during transcription (17Yu S.-L. Lee S.-K. Johnson R.E. Prakash L. Prakash S. Mol. Cell. Biol. 2003; 23: 382-388Google Scholar), or by promoting topoisomerase II-mediated double strand breaks (18Wilstermann A.M. Osheroff N. J. Biol. Chem. 2001; 276: 46290-46296Google Scholar). Of the repair pathways available to a cell (19Friedberg E.C. Nature. 2003; 421: 436-440Google Scholar, 20Christmann M. Tomicic M.T. Roos W.P. Kaina B. Toxicology. 2003; 193: 3-34Google Scholar), BER is the main pathway responsible for repairing AP sites in DNA. Initiation of BER is made possible by recognition of a damaged base by either a monofunctional or bifunctional DNA glycosylase, in addition to AP site recognition by APE. In monofunctional glycosylase-initiated BER (MFG-BER), a damaged or improper base is recognized and removed by enzymatic hydrolysis of the N-glycosyl bond resulting in the formation of an AP site. AP sites serve as a substrate for APE, which incises the DNA backbone immediately 5′ to the AP site via its 5′-endonuclease activity, producing a single strand break with a normal 3′-hydroxyl group and an abnormal 5′-deoxyribose 5-phosphate (dRP) residue (21Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis.2nd. Ed. American Society for Microbiology, Washington, D. C.1995: 208-270Google Scholar). DNA polymerase β (β-pol) inserts a new base followed by the coupled β-pol-mediated excision of the abnormal 5′-dRP, removal of which has been shown to be rate-limiting (22Srivastava D.K. Vande Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Google Scholar). In bifunctional glycosylaseinitiated BER, the damaged base is recognized and removed by a damage-specific DNA glycosylase followed by incision of the DNA backbone by the associated AP lyase activity, yielding a normal 5′-terminal deoxynucleoside-5′-phosphate residue and an abnormal 3′-terminal α,β-unsaturated aldehyde residue that must be processed prior to repair completion (21Friedberg E.C. Walker G.C. Siede W. DNA Repair and Mutagenesis.2nd. Ed. American Society for Microbiology, Washington, D. C.1995: 208-270Google Scholar). The removal of the abnormal 3′-blocking lesion by APE 3′-phosphodiesterase activity is believed to be rate-limiting (23Izumi T. Hazra T.K. Boldogh I. Tomkinson A.E. Park M.S. Ikeda S. Mitra S. Carcinogenesis. 2000; 21: 1329-1334Google Scholar); however, this rate-limiting role is controversial (24Cappelli E. Degan P. Frosina G. Carcinogenesis. 2000; 21: 1135-1141Google Scholar, 25Bogliolo M. Cappelli E. D'Osualdo A. Rossi O. Barbieri O. Kelley M.R. Frosina G. Anticancer Res. 2002; 22: 2797-2804Google Scholar, 26Cappelli E. D'Osualdo A. Bogliolo M. Kelley M.R. Frosina G. Environ. Mol. Mutagen. 2003; 42: 50-58Google Scholar). After APE recognition of AP sites, BER may proceed by one of two pathways: (i) β-pol-mediated single nucleotide insertion, similar to MFG-BER, or (ii) >1 nucleotide strand-displacement synthesis, required to process modified (i.e. reduced, oxidized) AP sites and involves components of the DNA replication machinery (27Demple B. DeMott M.S. Oncogene. 2002; 21: 8926-8934Google Scholar). Completion of BER requires the nick sealing activity of DNA ligase complexes (28Tomkinson A.E. Chen L. Dong Z. Leppard J.B. Levin D.S. Mackey Z.B. Motycka T.A. Prog. Nucleic Acids Res. Mol. Biol. 2001; 68: 151-164Google Scholar). A review of structural studies has proposed a model of BER requiring highly intimate, yet transient protein-protein interactions among BER enzymes to ensure proper damage repair, with APE being at the center of activity (29Wilson S.H. Kunkel T.A. Nat. Struct. Biol. 2000; 7: 176-178Google Scholar). For example, x-ray cross-complementing protein (XRCC1), a protein with no known enzymatic activity, functions as both a scaffold protein and modulator of BER via functional and physical interaction with APE, bridging the incision and nick-sealing steps of BER (30Vidal A.E. Boiteux S. Hickson I.D. Radicella J.P. EMBO J. 2001; 20: 6530-6539Google Scholar). APE has been shown also to interact with β-pol, recruiting it to the incised AP site and enhancing its rate-limiting dRPase activity (31Bennett R.A.O. Wilson III, D.M. Wong D. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7166-7169Google Scholar); this activity is also believed to be involved in the repair of oxidative base lesions (32Allinson S.L. Dianova I.I. Dianov G.L. EMBO J. 2001; 23: 6919-6926Google Scholar). The functional importance of β-pol in oxidative damage repair may be due to the interaction of APE with bifunctional DNA glycosylases responsible for recognizing and removing these lesions. For example, APE has been shown to stimulate 8-oxoguanine DNA glycosylase (OGG1) turnover and enhance its glycosylase activity while minimizing its associated AP lyase activity (33Hill J.W. Hazra T.K. Izumi T. Mitra S. Nucleic Acids Res. 2001; 29: 430-438Google Scholar, 34Vidal A.E. Hickson I.D. Boiteux S. Radicella J.P. Nucleic Acids Res. 2001; 29: 1285-1292Google Scholar), with XRCC1 accelerating this process (35Marsin S. Vidal A.E. Sossou M. Menissier-de Murcia J. Le Page F. Boiteux S. de Murcia G. Radicella J.P. J. Biol. Chem. 2003; 278: 44068-44074Google Scholar), thus eliminating a potentially rate-limiting step of APE and potentiating MFG-BER. Similar results have also been obtained with other bifunctional DNA glycosylases such as endonuclease III (hNTH1), responsible for recognition and removal of ring-saturated pyrimidines (36Marenstein D.R. Chan M.K. Altamirano A. Basu A.K. Boorstein R.J. Cunningham R.P. Teebor G.W. J. Biol. Chem. 2003; 278: 9005-9012Google Scholar). These studies, in addition to a recent mathematical model of BER throughput (37Sokhansanj B. Rodrigue G.R. Fitch J.P. Wilson III, D.M. Nucleic Acids Res. 2002; 30: 1817-1825Google Scholar), suggest a preference for β-pol-mediated MFG-BER in vivo. The objective of the research described in this study is to characterize in more detail the phenotype of an APE heterozygous knockout (Apex+/–) mouse reported previously (38Meira L.B. Devaraj S. Kisby G.E. Burns D.K. Daniel R.L. Hammer R.E. Grundy S. Jialal I. Friedberg E.C. Cancer Res. 2001; 61: 5552-5557Google Scholar), and to address the effect of APE haploinsufficiency on BER capacity. It is important to note that homozygous deletion of the APE gene (Apex–/–) is embryonic lethal, but heterozygous mice survive and are fertile (38Meira L.B. Devaraj S. Kisby G.E. Burns D.K. Daniel R.L. Hammer R.E. Grundy S. Jialal I. Friedberg E.C. Cancer Res. 2001; 61: 5552-5557Google Scholar, 39Ludwig D.L. MacInnes M.A. Takiguchi Y. Purtymun P.E. Henrie M. Flannery M. Meneses J. Pedersen R.A. Chen D.J. Mutat. Res. 1998; 409: 17-29Google Scholar, 40Xanthoudakis S. Smeyne R.J. Wallace J.D. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8919-8923Google Scholar). Because the sequences encoding both the DNA repair and redox regulatory activities of APE are disrupted in Apex–/– mice, it is unclear whether one or both of these activities are necessary for embryogenesis. Although the role of APE in the redox activation of p53 and other cellular transcription factors suggests its importance in signal transduction pathways, the embryonic lethality observed for APE and three other BER genes (β-pol, DNA ligase I, and XRCC1) suggests a critical role for BER during embryogenesis (41Friedberg E.C. Meira L.B. DNA Repair. 2003; 2: 501-530Google Scholar). Recent studies have implicated a role for p53 in the regulation of the BER pathway (42Smith M.L. Seo Y.R. Mutagenesis. 2002; 17: 149-156Google Scholar); therefore, it is inviting to suggest that APE repair activity in general, and perhaps APE redox regulatory activity in particular, is the reason for the embryonic lethality observed when APE is deficient. Here we present evidence that half the gene dosage of APE results in tissue-specific alterations in MFG-BER and suggest that APE redox activity, as opposed to repair per se, potentiates this phenotypic effect. Data obtained from Apex+/– mice may have relevant transnational implications because APE variants have been identified in the human population (43Hadi M.Z. Coleman M.A. Fidelis K. Mohrenweiser H.W. Wilson III, D.M. Nucleic Acids Res. 2000; 28: 3871-3879Google Scholar), and variants in DNA repair have been associated with increased risk for disease such as cancer (44de Boer J.G. Mutat. Res. 2002; 509: 201-210Google Scholar, 45Mohrenweiser H.W. Wilson III, D.M. Jones I.M. Mutat. Res. 2003; 526: 93-125Google Scholar). The experiments were performed on young (3–6 months) male C57BL/6-specific pathogen-free mice in accordance with the National Institutes of Health guidelines for the use and care of laboratory animals. The Wayne State University Animal Investigation Committee approved the animal protocol. Mice were maintained on a 12-h light/dark cycle and fed a standard lab diet and water ad libitum. The mice were sacrificed by cervical dislocation, and the organs to be studied were flash-frozen in liquid nitrogen and stored at –70 °C for later enzyme studies and Western blot analyses. Tissues for total RNA isolation and RT-PCR analysis were immediately homogenized in TRIzol® Reagent (Invitrogen) according to the manufacturer's protocol. The APE heterozygous knockout (Apex+/–) mice were developed in Friedberg's laboratory as described previously (38Meira L.B. Devaraj S. Kisby G.E. Burns D.K. Daniel R.L. Hammer R.E. Grundy S. Jialal I. Friedberg E.C. Cancer Res. 2001; 61: 5552-5557Google Scholar). In order to obtain the requisite number of animals for the study, the Apex+/– mice were inbred and maintained on a 12-h light/dark cycle and fed a standard lab diet and water ad libitum. The mice appeared normal, were fertile, and there was no retardation in food intake, weight gain, or growth rate; however, it was observed that pups were not produced in expected Mendelian ratios, with heterozygote births predominating, similar to previous observations (39Ludwig D.L. MacInnes M.A. Takiguchi Y. Purtymun P.E. Henrie M. Flannery M. Meneses J. Pedersen R.A. Chen D.J. Mutat. Res. 1998; 409: 17-29Google Scholar). A three-primer PCR strategy was employed to genotype the animals generated by Apex+/– intercrosses as described previously (38Meira L.B. Devaraj S. Kisby G.E. Burns D.K. Daniel R.L. Hammer R.E. Grundy S. Jialal I. Friedberg E.C. Cancer Res. 2001; 61: 5552-5557Google Scholar). The level of APE mRNA was measured using RT-PCR analysis using AccessQuick™ RT-PCR System (Promega, Madison, WI) according to the manufacturer's protocol. Total cellular RNA was isolated from selected tissues using TRIzol® Reagent (Invitrogen), and RNA concentration was determined by measuring UV absorption at 260/280 nm. Oligonucleotide primers specific for the mouse Apex gene (forward, 5′-CTCAAGATATGCTCCTGGAA-3′; reverse, 5′-GGTATTCCAGTCTTACCAGA-3′) were designed using GeneFisher Interactive Primer Design Tool (Bielefeld, Germany) and synthesized by Sigma-Genosys (The Woodlands, TX). RT-PCR thermal cycling conditions were as follows: 48 °C for 45 min, 1 cycle; 95 °C for 2 min, 1 cycle; 95 °C for 1 min, 52 °C for 1 min, 70 °C for 2 min, 22 cycles; and 70 °C for 5 min, 1 cycle. The 350-bp RT-PCR product was stained with ethidium bromide and analyzed on a 2% agarose gel. Intensity of the bands was detected and quantified using a ChemiImager™ system (AlphaInnotech, San Leandro, CA) and expressed as the integrated density value (I.D.V) per μg of RNA used per reaction. Data were normalized based on the amount of β-actin present in each sample. Isolation of crude nuclear extract was accomplished using Sigma CelLytic™ NuCLEAR™ extraction kit (Sigma). All samples and tubes were handled and chilled on ice, and all solutions were made fresh according to the manufacturer's protocol. Resultant nuclear extracts were dialyzed against 1 liter of dialysis buffer (20 mm Tris-HCl, pH 8.0; 100 mm KCl; 10 mm NaS2O5; 0.1 mm DTT; 0.1 mm phenylmethylsulfonyl fluoride; 1 μg/ml pepstatin A) for 4–6 h at 4 °C using Slide-A-Lyzer® minidialysis units (Pierce). Protein concentrations were determined using Protein Assay Kit I (Bio-Rad). Western analysis was performed using 200 μg of crude nuclear extract isolated from selected tissues according to standard protocol. Protein levels were determined using manufacturer recommended dilutions of monoclonal antisera developed against mouse APE/REF-1 (Novus Biologicals, Littleton, CO), and monoclonal antisera developed against rat β-pol (Ab-1 Clone 18S, NeoMarkers, Fremont, CA). The bands were detected and quantified using a ChemiImager™ system (AlphaInnotech, San Leandro, CA) after incubation in SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Data are expressed as the I.D.V. of the band per μg of protein loaded. The 5′-endonuclease activity of APE was analyzed using a quantitative in vitro assay that measures the incision of a 26-mer duplex oligonucleotide substrate containing a synthetic tetrahydrofuran (F) AP site (upper strand, 5′-AATTCACCGGTACCFTCTAGAATTCG-3′; lower strand, 5′-CGAATTCTAGAGGGTACCGGTGAATT-3′) as described previously (46Wilson III, D.M. Takeshita M. Grollman A.P. Demple B. J. Biol. Chem. 1995; 270: 16002-16007Google Scholar). Briefly, 2.5 pmol of radio-end-labeled and purified duplex AP DNA substrate was incubated with 100 ng of crude nuclear extract-selected tissues in a 10-μl reaction mixture containing 50 mm Hepes, pH 7.5; 50 mm KCl; 10 mm MgCl2; 2mm DTT; 1 μg/ml BSA; and 0.05% Triton X-100. The reaction mixtures were incubated for 15 min at 37 °C and stopped by the addition of 50 mm EDTA. Assay products (5 μl) were added to 15 μl of loading dye (95% formamide, 5% glycerol, 10 mg of xylene cyanol, 10 mg of bromphenol blue) and heated at 95 °C for 5 min. Aliquots (3 μl) were run on a 15% denaturing 19:1 acrylamide/bisacrylamide gel (SequaGel® Sequencing System, National Diagnostics, Atlanta, GA) at 55 °C, soaked in fixing solution (10% glacial acetic acid; 10% methanol), wrapped in Saran wrap, and exposed to a Molecular Imaging Screen (Bio-Rad). Endonuclease activity (presence of a 14-mer band) was visualized and quantified using a Molecular Imager® System (Bio-Rad) by calculating the relative amount of the 14-mer oligo product with the unreacted 26-mer substrate (product/(product + substrate)). Data are expressed as machine counts per ng of protein. The principle of this assay is to measure MFG-BER activity. Radio end-labeled and purified 30-bp oligonucleotides (upper strand, 5′-ATATACCGCGGUCGGCCGATCAAGCTTATTdd-3′; lower strand, 3′-ddTATATGGCGCCGGCCGGCTAGTTCGAATAA-5′) containing a G:U mismatch and an HpaII restriction site (CCGG) were incubated in a reaction mixture (100 mm Tris-HCl, pH 7.5, 5 mm MgCl2; 1 mm DTT, 0.1 mm EDTA, 2 mm ATP, 0.5 mm NAD, 20 μm dNTPs, 5 mm diTrisphosphocreatine, 10 units of creatine phosphokinase) with 50 μg of crude nuclear extract isolated from selected tissues. The reaction mixtures were incubated for 30 min at 37 °C, followed by 5 min at 95 °C to stop the reaction. The duplex oligonucleotides were allowed to reanneal for 1 h at room temperature before being briefly centrifuged to pellet the denatured proteins. Repair of the G:U mismatch to a correct G:C base pair was determined via treatment of the duplex oligonucleotide with 20 units of HpaII (Promega, Madison, WI) for 1 h at 37 °C and analysis by electrophoresis on a 20% denaturing 19:1 acrylamide/bisacrylamide gel (SequaGel® Sequencing System, National Diagnostics, Atlanta, GA). Repair activity (presence of a 16-mer band) was visualized and quantified using a Molecular Imager® System (Bio-Rad) by calculating the ratio of the 16-mer product with the 30-mer substrate (product/substrate). Data are expressed as machine counts per μg of protein. The DNA binding ability of nuclear extracts isolated from selected tissues was determined using electrophoretic mobility shift assays (EMSAs). Nuclear extracts (20 μg) were incubated with 4× reaction buffer (final concentrations: 50 mm Hepes, pH 8.0; 100 mm NaCl; 10 mm EDTA; 1 mm DTT; 9.5% glycerol v/v) plus 1 μg of BSA and 2 μg of poly(dI-dC) for 5 min at room temperature. A radio-end-labeled and purified oligonucleotide probe containing an AP site (0.0125 pmol) was added to the reaction mix and incubated for 30 min at room temperature. Negative controls (all components except nuclear extract) were included in all experiments. In competitive assays, 100× molar excess of unlabeled DNA was added to the reaction mixture. The protein-DNA complex was resolved on a 5% non-denaturing polyacrylamide gel in 0.5× TBE buffer. Reaction products were visualized and quantified using a Molecular Imager® System (Bio-Rad). TEMPO Extraction of DNA—DNA for the aldehyde-reactive probe slot blot (ASB) assay was extracted according to the method described by Hofer and Moller (47Hofer T. Moller L. Chem. Res. Toxicol. 1998; 11: 882-887Google Scholar) with some modifications. This method minimizes artifactual DNA damage by using 20 mm TEMPO in all solutions and reagents and by minimizing heat treatment of DNA. Briefly, tissue was homogenized in ice-cold PBS and centrifuged (2000 × g at 4 °C for 5 min). Resultant supernatant was decanted and pellet resuspended in lysis buffer (Applied Biosystems, Foster City, CA). Proteinase K (30 units, Ambion, Austin, TX) was added, and samples were incubated overnight at 4 °C, followed by phenol/chloroform and Sevag (chloroform/isoamyl alcohol, 24:1) extraction. Extracted DNA was precipitated using 7.5% 4 m NaCl and 2 volumes of 100% cold ethanol and centrifuged (2000 × g at 4 °C for 5 min). Resultant pellet was washed in 70% ethanol and resuspended in ice-cold PBS and rehydrated at 4 °C. The samples were incubated with RNase A (2 μg) and RNase T1 (1,000 units, Ambion, Austin, TX) for 30 min at 37 °C, and resultant DNA was cold ethanol-precipitated, resuspended in deionized water at 4 °C, and stored at –70 °C. Gravity Tip Extraction of DNA—DNA for the random oligonucleotide primed synthesis (ROPS) assay was isolated using Qiagen (Valencia, CA) gravity tip columns as described in the manufacturer's protocol. This method generates large fragments of DNA (up to 150-kb) while minimizing shearing. ASB—The ASB assay was carried out as described previously (14Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225Google Scholar) with slight modifications. TEMPO-extracted DNA (8 μg) was incubated in 30 μl of PBS with 2 mm aldehyde-reactive probe (ARP) (Dojindo Laboratories, Kumamoto, Japan) at 37 °C for 10 min. DNA was cold ethanol-precipitated (as described above) and resuspended in 1× TE buffer overnight at 4 °C. DNA was heat-denatured at 100 °C for 5 min, quickly chilled on ice, and mixed with equal amount of 2 m ammonium acetate. DNA was immobilized on a nitrocellulose membrane (Schleicher & Schuell) by using a Invitrogen Filtration Manifold system. The membrane was washed in 5× SSC for 15 min at 37 °C and then baked under vacuum at 80 °C for 30 min. The dried membrane was incubated in a hybridization buffer (final concentrations: 20 mm Tris, pH 7.5; 0.1 m NaCl; 1 mm EDTA; 0.5% casein w/v; 0.25% BSA w/v; 0.1% Tween 20 v/v) for 30 min at room temperature. The membrane was then incubated in the same hybridization buffer containing 100 μl of streptavidin-conjugated horseradish peroxidase (BioGenex, San Ramon, CA) at room temperature for 45 min. Following incubation in horseradish peroxidase, the membrane was washed three times for 5 min each at 37 °C in TBS, pH 7.5 (final concentrations: 0.26 m NaCl; 1 mm EDTA; 20 mm Tris, pH 7.5; 0.1% Tween 20). The membrane was incubated in ECL (Pierce) for 5 min at room temperature and visualized using a ChemiImager™ system (AlphaInnotech, San Leandro, CA). Standards containing known amounts of AP sites (14Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225Google Scholar) were used to determine the relative level of AP sites in liver DNA. Data are expressed as number of AP sites per 106 nucleotides. ROPS—The relative number of 3′-OH group-containing DNA strand breaks was quantified using a Klenow(exo–) incorporation assay based on the ability of Klenow to initiate DNA synthesis from 3′-OH ends of single strand DNA (48Basnakian A.G. James S.J. DNA Cell Biol. 1996; 15: 255-262Google Scholar). Gravity tip extracted DNA (0.25 μg) was heat-denatured at 100 °C for 5 min and added to the Klenow reaction buffer (0.5 mm dTTP, 0.5 mm dGTP, and 0.5 mm dATP; 0.33 μm dCTP, 1 μl of Klenow(exo–)) with 10× Klenow buffer per manufacturer's protocol (New England Biolabs, Beverly, MA) and 5 μCi of [α-32P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences). Reaction mixtures were incubated at 16 °C for 30 min, and the reaction was stopped with the addition of 25 μl of 12.5 mm EDTA, pH 8.0. Samples (5 μl) were spotted onto scored and numbered Whatman DE81 paper and allowed to air dry. The spotted chromatography paper was washed five times for 5 min in 0.5 m Na2HPO4 (dibasic) followed by a brief rinse in deionized water two times and then allowed to air-dry. Paper was cut and placed into scintillation vials with 2.5 ml of ScintiVerse mixture (Fisher). Incorporation of [α-32P]dCTP was quantified using a Packard scintillation counter. Statistical significance between means was determined using analysis of variance followed by the Fisher's least significant difference test where appropriate (49Sokal R.R. Rohlf F.J. Biometry. W. H. Freeman & Co., New York1981: 169-176Google Scholar). A p value less than 0.05 was considered statistically significant. In order to elucidate the molecular effects of gene-targeted disruption of the mouse APE gene (Apex), our laboratory has characterized in detail a transgenic "knockout" mouse containing a heterozygous deletion of the APE gene (Apex+/–). As reported previously, the Apex–/– mice are embryonic lethal, whereas the Apex+/– animals are fertile, appear normal, and exhibit reduced APE mRNA and APE protein levels in mouse embryonic fibroblasts and brain cells as compared with their Apex+/+ counterparts (38Meira L.B. Devaraj S. Kisby G.E. Burns D.K. Daniel R.L. Hammer R.E. Grundy S. Jialal I. Friedberg E.C. Cancer Res. 2001; 61: 5552-5557Google Scholar). In a series of experiments, we were able to confirm whether APE heterozygosity would cause these mice to exhibit haploinsufficiency with respect to APE in brain, liver, and testes. In order to determine whether loss of a functional allele of APE would result in reduced expression of this gene, we have quantified the expression of APE via RT-PCR analysis. By using total RNA isolated from liver, we observed a 40–50% decrease in APE mRNA (Fig. 1A) in Apex+/– mice as compared with their normal, wild-type (Apex+/+) counterparts. Because differences in APE mRNA may not necessarily reflect differences in APE protein levels, we also measured APE protein levels using Western blot analysis. We show a corresponding 40–50% decrease in APE protein in liver (Fig. 1B). To confirm that changes in APE expression (i.e. changes in mRNA and protein levels) result in changes in APE enzymatic activity, we measured APE 5′-endonuclease activity using a 26-bp oligonucl
AP endonuclease
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AP endonuclease
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Phosphodiester bond
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AP endonuclease
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The integrity of cellular genome is continuously challenged by endogenous and exogenous DNA damaging agents. If DNA damage is not removed in a timely fashion the replisome may stall at DNA lesions, causing fork collapse and genetic instability. Base excision DNA repair (BER) is the most important pathway for the removal of oxidized or mono-alkylated DNA. While the main components of the BER pathway are well defined, its regulatory mechanism is not yet understood. We report here that the splicing factor ISY1 enhances apurinic/apyrimidinic endonuclease 1 (APE1) activity, the multifunctional enzyme in BER, by promoting its 5'-3' endonuclease activity. ISY1 expression is induced by oxidative damage, which would provide an immediate up-regulation of APE1 activity in vivo and enhance BER of oxidized bases. We further found that APE1 and ISY1 interact, and ISY1 enhances the ability of APE1 to recognize abasic sites in DNA. Using purified recombinant proteins, we reconstituted BER and demonstrated that ISY1 markedly promoted APE1 activity in both the short- and long-patch BER pathways. Our study identified ISY1 as a regulator of the BER pathway, which would be of physiological relevance where suboptimal levels of APE1 are present. The interaction of ISY1 and APE1 also establishes a connection between DNA damage repair and pre-mRNA splicing.
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Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins that work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases resulting in abasic sites. Many DNA glycosylases are product inhibited by binding to the abasic site more avidly than the damaged base. Traditionally apurinic/apyrimidinic endonuclease, APE1, was believed to help turnover the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three-to-five-fold. Moreover, we have shown UV-DDB can assist chromatin decompaction facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER.
MUTYH
AP endonuclease
XRCC1
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Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.
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Adenine-DNA glycosylase MutY of Escherichia coli catalyzes the cleavage of adenine when mismatched with 7,8-dihydro-8-oxoguanine (GO), an oxidatively damaged base. The biological outcome is the prevention of C/G→A/T transversions. The molecular mechanism of base excision repair (BER) of A/GO in mammals is not well understood. In this study we report stimulation of mammalian adenine-DNA glycosylase activity by apurinic/apyrimidinic (AP) endonuclease using murine homolog of MutY (Myh) and human AP endonuclease (Ape1), which shares 94% amino acid identity with its murine homolog Apex. After removal of adenine by the Myh glycosylase activity, intact AP DNA remains due to lack of an efficient Myh AP lyase activity. The study of wild-type Ape1 and its catalytic mutant H309N demonstrates that Ape1 catalytic activity is required for formation of cleaved AP DNA. It also appears that Ape1 stimulates Myh glycosylase activity by increasing formation of the Myh–DNA complex. This stimulation is independent of the catalytic activity of Ape1. Consequently, Ape1 preserves the Myh preference for A/GO over A/G and improves overall glycosylase efficiency. Our study suggests that protein–protein interactions may occur in vivo to achieve efficient BER of A/GO.
AP endonuclease
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Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins which work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases, resulting in abasic sites. Many DNA glycosylases are product-inhibited by binding to the abasic site more avidly than the damaged base. Traditionally, apurinic/apyrimidinic endonuclease 1, APE1, was believed to help turn over the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory, we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three- and five-fold. Moreover, we have shown that UV-DDB can assist chromatin decompaction, facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER.
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Bcl2 not only prolongs cell survival but also suppresses the repair of abasic (AP) sites of DNA lesions. Apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in the repair of AP sites via the base excision repair pathway. Here we found that Bcl2 down-regulates APE1 endonuclease activity in association with inhibition of AP site repair. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus and interaction with APE1, which requires all of the BH domains of Bcl2. Deletion of any of the BH domains from Bcl2 abrogates the ability of Bcl2 to interact with APE1 as well as the inhibitory effects of Bcl2 on APE1 activity and AP site repair. Overexpression of Bcl2 in cells reduces formation of the APE1·XRCC1 complex, and purified Bcl2 protein directly disrupts the APE1·XRCC1 complex with suppression of APE1 endonuclease activity in vitro. Importantly, specific knockdown of endogenous Bcl2 by RNA interference enhances APE1 endonuclease activity with accelerated AP site repair. Thus, Bcl2 inhibition of AP site repair may occur in a novel mechanism by down-regulating APE1 endonuclease activity, which may promote genetic instability and tumorigenesis. Bcl2 not only prolongs cell survival but also suppresses the repair of abasic (AP) sites of DNA lesions. Apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in the repair of AP sites via the base excision repair pathway. Here we found that Bcl2 down-regulates APE1 endonuclease activity in association with inhibition of AP site repair. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus and interaction with APE1, which requires all of the BH domains of Bcl2. Deletion of any of the BH domains from Bcl2 abrogates the ability of Bcl2 to interact with APE1 as well as the inhibitory effects of Bcl2 on APE1 activity and AP site repair. Overexpression of Bcl2 in cells reduces formation of the APE1·XRCC1 complex, and purified Bcl2 protein directly disrupts the APE1·XRCC1 complex with suppression of APE1 endonuclease activity in vitro. Importantly, specific knockdown of endogenous Bcl2 by RNA interference enhances APE1 endonuclease activity with accelerated AP site repair. Thus, Bcl2 inhibition of AP site repair may occur in a novel mechanism by down-regulating APE1 endonuclease activity, which may promote genetic instability and tumorigenesis. Apurinic/apyrimidinic (AP) 2The abbreviations used are:AP siteapurinic/apyrimidinic or abasic siteAPE1apurinic/apyrimidinic endonuclease 1NNKnitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1y-butanoneBERbase excision repairBHBcl2 homologysiRNAsmall interfering RNAWTwild typePBSphosphate-buffered salineCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidARPaldehyde reactive probe. or abasic sites are the most common form of DNA damage with about 20,000–50,000 sites produced in each cell/day (1Kelley M. Parsons S. Antioxid. Redox Signal. 2001; 3: 671-683Crossref PubMed Scopus (91) Google Scholar, 2Lau J. Weatherdon K. Skalski V. Hedley D. Br. J. Cancer. 2004; 91: 1166-1173Crossref PubMed Scopus (64) Google Scholar). AP sites can result from spontaneous and chemically initiated hydrolysis through various conditions, including ionizing radiation, UV irradiation, oxidative stress, and exposure to cigarette smoking (1Kelley M. Parsons S. Antioxid. Redox Signal. 2001; 3: 671-683Crossref PubMed Scopus (91) Google Scholar, 2Lau J. Weatherdon K. Skalski V. Hedley D. Br. J. Cancer. 2004; 91: 1166-1173Crossref PubMed Scopus (64) Google Scholar, 3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar, 4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Human apurinic/apyrimidinic endonuclease 1 (APE1) is a major constituent of the base excision repair (BER) pathway of AP sites of DNA lesions (3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar). Additionally, APE1 is also named as redox effector factor-1 (1Kelley M. Parsons S. Antioxid. Redox Signal. 2001; 3: 671-683Crossref PubMed Scopus (91) Google Scholar) because of its redox abilities on different redox-regulated transcription factors (3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar, 5Xanthoudakis S. Miao G. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 23-27Crossref PubMed Scopus (320) Google Scholar). Two activities of this molecule are split into two functionally independent domains of the protein itself: the N terminus is principally devoted to the redox activity, whereas the C terminus exerts enzymatic activity on the repair of AP sites of DNA lesions (3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar, 5Xanthoudakis S. Miao G. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 23-27Crossref PubMed Scopus (320) Google Scholar). APE1 specifically binds to abasic sites and cuts the 5′ phosphodiester bond with its endonuclease activity to produce a DNA primer with 3′ hydroxyl end, which is a required step in the BER repair pathway (3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar). Therefore, APE1 is an essential endonuclease and plays a central role in the repair of AP sites of DNA lesions. apurinic/apyrimidinic or abasic site apurinic/apyrimidinic endonuclease 1 nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1y-butanone base excision repair Bcl2 homology small interfering RNA wild type phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid aldehyde reactive probe. Bcl2, a major cellular oncogenic protein, plays pivotal roles in enhancing cell survival, retarding G1/S cell cycle transition, and attenuating DNA repair (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 6Deng X. Gao F. Flagg T. Anderson J. May W.S. Mol. Cell. Biol. 2006; 26: 4421-4434Crossref PubMed Scopus (116) Google Scholar, 7Deng X. Gao F. Flagg T. May W.S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 153-158Crossref PubMed Scopus (133) Google Scholar). Because overexpression of Bcl2 results in lymphomagenesis in transgenic mice, this suggests that Bcl2, in addition to its survival activity, may also potentially have an oncogenic property (8Linette G.P. Hess J.L. Sentman C.L. Korsmeyer S.J. Blood. 1995; 86: 1255-1260Crossref PubMed Google Scholar). However, the mechanism(s) by which Bcl2 facilitates oncogenesis is not fully understood. The oncogenic effect of Bcl2 may result from its multiple cellular properties. Bcl2 was originally discovered as a gene product at the chromosomal breakpoint of t(14;18) (9Tsujimoto Y. Cossman J. Jaffe E. Croce C.M. Science. 1985; 229: 1390-1393Crossref PubMed Scopus (837) Google Scholar), which may play a role in genetic instability and tumor development by impeding DNA repair. It has been reported that Bcl2 can enhance benzene metabolite-induced DNA damage and mutagenesis in human promyelocytic HL60 cells (10Kuo M. Shiah S. Wang C. Chuang S. Mol. Pharmacol. 1999; 55: 894-901PubMed Google Scholar). Overexpression of Bcl2 not only attenuates the nucleotide excision repair capacity and DNA replication in UV-irradiated HL60 cells (11Liu Y. Naumovski L. Hanawalt P. Cancer Res. 1997; 57: 1650-1653PubMed Google Scholar) but also inhibits γ-ray-induced homologous recombination repair pathways (12Saintigny Y. Dumay A. Lambert S. Lopez B. EMBO J. 2001; 20: 2596-2607Crossref PubMed Scopus (82) Google Scholar). Bcl2 can also suppress DNA mismatch repair by inhibiting E2F transcriptional activity (13Youn C.K. Cho H. Kim S. Kim H. Kim M. Chang I. Lee J. Chung M. Hahm K. You H. Nat. Cell Biol. 2005; 7: 137-147Crossref PubMed Scopus (67) Google Scholar). Intriguingly, our recent findings reveal that Bcl2 suppression of DNA mismatch repair occur in a mechanism by directly regulating the heterodimeric hMSH2·hMSH6 complex, which leads to enhanced mutagenesis (14Hou Y. Gao F. Wang Q. Zhao J. Flagg T. Zhang Y. Deng X. J. Biol. Chem. 2007; 282: 9279-9287Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Thus, the oncogenic activity of Bcl2 may result from its inhibitory effects on multiple DNA repair pathways. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that can induce cellular DNA damage including AP sites of DNA lesions (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 15Jorquera R. Castonguay A. Schuller H. Carcinogenesis. 1994; 15: 389-394Crossref PubMed Scopus (29) Google Scholar, 16Mijal R. Thomson N. Fleischer N. Pauly G. Moschel R. Kanugula S. Fang Q. Pegg A. Peterson L. Chem. Res. Toxicol. 2004; 17: 424-434Crossref PubMed Scopus (58) Google Scholar). NNK-induced AP sites of DNA lesions, if unrepaired or repaired incorrectly, could be mutagenic, which may lead to mutations and chromosomal breaks with malignant transformation. We previously discovered that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, the mechanism(s) is not fully understood. Here we found that Bcl2 not only directly interacts with APE1 but also inhibits its endonuclease activity, which leads to suppression of AP site repair. Materials—Bcl2, APE1, XRCC1, and tubulin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). NNK was purchased from Toronto Research Chemicals (Toronto, Canada). Purified recombinant WT, ΔBH1, ΔBH2, ΔBH3, and ΔBH4 Bcl2 mutant proteins were obtained from ProteinX Lab (San Diego, CA). Purified recombinant APE1 protein was purchased from Abnova Corporation (Taipei, Taiwan). Synthetic human Bcl2 siRNA (sense strand sequence, GGAUCCAGGAUAACGGAGGTT) was obtained from Santa Cruz Biotechnology. All of the reagents used were obtained from commercial sources unless otherwise stated. Generation of Various Bcl2 Deletion Mutants—To create ΔBH1, ΔBH2, ΔBH3, and ΔBH4 Bcl2 deletion mutants, the 5′ phosphorylated mutagenic primers for various precise deletion mutants were synthesized as follow: ΔBH1, 5′-GGA CGC TTT GCC ACG GTG GTG GAG GTG GAG AGC GTC AAC AGG GAG ATG-3′; ΔBH2, 5′-GAG TAC CTG AAC CGG CAT CTG CAC CGA CCT CTG TTT GAT TTC TCC TGG-3′; ΔBH3, 5′-GCG CTC AGC CCT GTG CCA CCT GTG GAC TTC GCA GAG ATG TCC AGT CAG-3′; ΔBH4, 5′-GGA AGG ATG GCG CAA GCC GGG AGA GCT GGA GAT GCG GAC GCG GCG CCC CTG-3′. The WT-Bcl2/pUC19 construct was used as the target plasmid that contains a unique NdeI restriction site for selection against the unmutated plasmid. The NdeI selection primer is: 5′-GAG TGC ACC ATG GGC GGT GTG AAA-3′. Various Bcl2 BH deletion mutants were created by using a mutagenesis kit (Clontech) according to the manufacturer's instructions and confirmed by sequencing of the cDNA. The WT and various Bcl2 deletion mutants were then cloned into the pCIneo (Promega) mammalian expression vector. Cell Lines, Plasmids, and Transfections—Various human lung cancer cells were maintained as previously described (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The pCIneo plasmid containing each Bcl2 mutant cDNA was transfected into H1299 cells using LipfectAMINE™2000 (Invitrogen). Clones stably expressing WT or each of the Bcl2 deletion mutants were selected in a medium containing G418 (0.6 mg/ml). The expression levels of exogenous Bcl2 were analyzed by Western blot analysis. Three separate clones for each mutant expressing similar amounts of exogenous Bcl2 were selected for further analysis. Preparation of Cell Lysates—Cells were washed with 1× PBS and resuspended in ice-cold 1% CHAPS lysis buffer (1% CHAPS, 50 mm Tris, pH 7.6, 120 mm NaCl, 1 mm EDTA, 1 mm Na3VO4, 50 mm NaF, and 1 mm β-mercaptoethanol) with a mixture of protease inhibitors (Calbiochem). The cells were lysed by sonication and centrifuged at 14,000 × g for 10 min at 4 °C. The resulting supernatant was collected as the total cell lysate and used for protein analysis or co-immunoprecipitation as described (6Deng X. Gao F. Flagg T. Anderson J. May W.S. Mol. Cell. Biol. 2006; 26: 4421-4434Crossref PubMed Scopus (116) Google Scholar). Subcellular Fractionation—Subcellular fractionation was performed as described previously (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Briefly, the cells (2 × 107) were washed once with cold 1× PBS and resuspended in isotonic mitochondrial buffer (210 mm mannitol, 70 mm sucrose, 1 mm EGTA, 10 mm Hepes, pH 7.5) containing protease inhibitor mixture set I (Calbiochem). The resuspended cells were homogenized with a polytron homogenizer operating for four bursts of 10 s each at a setting of 5 and then centrifuged at 2000 × g for 3 min to pellet the nuclei and unbroken cells. The supernatant was centrifuged at 13,000 ×g for 10 min to pellet mitochondria. The mitochondria was washed with mitochondrial buffer, resuspended with 1% Nonidet P-40 lysis buffer, rocked for 60 min, and then centrifuged at 17,530 × g for 10 min at 4 °C. The supernatant containing mitochondrial proteins was collected. For nuclear fractionation, the cells were washed with 1× PBS and suspended in 2 ml of Buffer A (10 mm Tris-HCl, pH 7.4, 10 mm NaCl, 3 mm MgCl2, 0.03% Nonidet P-40 with fresh protease inhibitor mixture set I). The samples were incubated on ice until more than 95% of cells could be stained by trypan blue. The samples were then centrifuged at 500 × g at 4 °C for 5 min. The resulting nuclear pellet was washed with Buffer B (50 mm NaCl, 10 mm Hepes, pH 8.0, 25% glycerol, 0.1 mm EDTA, 0.5 mm spermidine, 0.15 mm spermine) and then resuspended in 150 μl of Buffer C (350 mm NaCl, 10 mm Hepes, pH 8.0, 25% glycerol, 0.1 mm EDTA, 0.5 mm spermidine, 0.15 mm spermine) and rocked at 4 °C for 30 min. After centrifugation (14, 000 × g) at 4 °C, the supernatant (nuclear fraction) was collected. Protein (100 μg) from each fraction was subjected to SDS-PAGE and analyzed by Western blotting using a Bcl2 antibody. Immunofluorescence Staining—The cell were washed with 1× PBS, fixed with cold methanol and acetone (1:1) for 5 min, and then blocked with 10% normal rabbit serum for 20 min at room temperature. The cells were incubated with a rabbit Bcl2 primary antibody for 90 min. After washing, the samples were incubated with Alexa 594 (red)-conjugated anti-rabbit secondary antibodies for 60 min. The cells were washed with 1× PBS and observed under a fluorescent microscope (Zeiss). Pictures were taken of each sample. AP Oligonucleotide Assay for APE1 Endonuclease Activity—A 26-mer oligonucleotide (IDT Technologies, Coralville, IA) containing a tetrahydrofuran (F) residue at position 15 was used as the APE1 substrate as described (1Kelley M. Parsons S. Antioxid. Redox Signal. 2001; 3: 671-683Crossref PubMed Scopus (91) Google Scholar, 2Lau J. Weatherdon K. Skalski V. Hedley D. Br. J. Cancer. 2004; 91: 1166-1173Crossref PubMed Scopus (64) Google Scholar). Following 32P labeling, the oligonucleotides were purified using a G25 column and then annealed to a complementary oligonucleotide. Assays using nuclear extract isolated from cells or purified APE1 protein were performed in a 20-μl reaction volume containing 2.5 pmol of labeled double-stranded F oligonucleotide, in 50 mm Hepes, 50 mm KCl, 10 mm MgCl2, 2 mm dithiothreitol, 1 μg/ml bovine serum albumin, 0.05% Triton X-100, pH 7.5. The reactions were allowed to proceed for 15 min in a 37 °C water bath and stopped by adding an equal volume of 96% formamide, 10 mm EDTA, and bromphenol blue. 10 μl of this 40-μl sample was separated with a 20% polyacrylamide gel containing 7 m urea. APE1 endonuclease activity was analyzed by autoradiography. AP Site Counting in Genomic DNA—Genomic DNA was purified using a DNA isolation kit (Dojindo Molecular Technologies, Inc., Gaithersburg, MD). The number of AP sites was assessed using a DNA damage quantification (AP site counting) kit according to the manufacturer's instructions (Dojindo Molecular Technologies, Inc.). Aldehyde reactive probe (ARP) reagent (N′-aminooxymethylcarbonylhydrazino-d-biotin) can react specifically with an aldehyde group that is the open ring form of the AP sites. After treating DNA containing AP sites with ARP reagent, AP sites are tagged with biotin residues. By using an excess amount of ARP, all AP sites can be converted to biotin-tagged AP sites. Standard ARP DNA and purified ARP-labeled sample genomic DNA was fixed on a 96-well plate with DNA binding solution. Then the number of AP sites in the sample DNA was determined by the biotin-avidin-peroxidase assay. The absorbance of the samples was analyzed using a microplate reader with a 650-nm filter. Each experiment was repeated three times, and the data represent the means ± S.D. of three determinations. Bcl2 Silence—This is a technique for down-regulating the expression of a specific gene in living cells by introducing a short homologous double-stranded RNA. H460 cells expressing high levels of endogenous Bcl2 were transfected with Bcl2 siRNA (10 nm) using LipofectAMINE™ 2000. A control siRNA (nonhomologous to any known gene sequence) was used as a negative control. The levels of Bcl2 expression were analyzed by Western blotting. Specific silencing of the targeted Bcl2 gene was confirmed by at least three independent experiments. Expression of Endogenous Bcl2 Is Associated with Decreased APE1 Endonuclease Activity in Various Human Lung Cancer Cells—APE1 is a multifunctional DNA repair enzyme that mainly functions as an abasic endonuclease in the BER pathway (1Kelley M. Parsons S. Antioxid. Redox Signal. 2001; 3: 671-683Crossref PubMed Scopus (91) Google Scholar, 2Lau J. Weatherdon K. Skalski V. Hedley D. Br. J. Cancer. 2004; 91: 1166-1173Crossref PubMed Scopus (64) Google Scholar, 3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar). Intriguingly, APE1 is widely expressed in both small cell lung cancer and nonsmall cell lung cancer cells (Fig. 1A), suggesting that APE1 may play a role in regulating DNA repair in human lung cancer cells. Bcl2, a major antiapoptotic and/or oncogenic protein, is found to co-express with APE1 in H69 and H460 but not in other lung cancer cells tested (Fig. 1A). We previously discovered that Bcl2 can suppress the repair of abasic sites of DNA lesions (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). However, the mechanism(s) remains enigmatic. To test whether endogenous expression of Bcl2 may potentially regulate APE1 function, AP endonuclease activity was measured in various lung cancer cells. A 26-mer 32P-labeled, AP site mimetic, double-stranded F oligonucleotide was used as an APE1 substrate as described (1Kelley M. Parsons S. Antioxid. Redox Signal. 2001; 3: 671-683Crossref PubMed Scopus (91) Google Scholar, 2Lau J. Weatherdon K. Skalski V. Hedley D. Br. J. Cancer. 2004; 91: 1166-1173Crossref PubMed Scopus (64) Google Scholar). As shown in Fig. 1B, the cleaved product should be observed as a 14-mer fragment that will indicate the AP endonuclease repair activity. By contrast, the uncleaved oligonucleotide correlating to no activity should be observed as a 26-mer fragment. Thus, the AP endonuclease activity was determined by the amount of cleaved product (14-mer). 32P-labeled 26-mer AP site mimetic oligonucleotides were incubated with nuclear extract isolated from various human lung cancer cells. Decreased levels of AP endonuclease activity (i.e. smaller amount of cleaved 14-mer products and greater amount of uncleaved 26-mer oligonucleotides) were observed in both H460 and H69 cells that express high levels of endogenous Bcl2 as compared with the other cell lines that express undetectable levels of Bcl2 (Fig. 1C), suggesting that Bcl2 may play a negative role in regulating APE1 endonuclease activity. Overexpression of Exogenous Bcl2 Down-regulates APE1 Activity and Potently Suppresses AP Site Repair—To directly test effects of Bcl2 on APE1 function and AP site repair, Bcl2 was stably overexpressed in H1299 cells that do not express detectable levels of endogenous Bcl2. A majority of the 26-mer oligonucleotides was cleaved into 14-mer products after incubation with the nuclear extract isolated from vector-only control cells, indicating a strong AP endonuclease activity in vector-only H1299 cells (Fig. 2, A and B). In contrast, overexpression of Bcl2 prevents the 26-mer oligonucleotides from cleavage (Fig. 2, A and B). These findings suggest that Bcl2 suppresses APE1 endonuclease activity. Similar results were obtained from three independent clones expressing similar levels of exogenous Bcl2, indicating that these findings are reliable. Because APE1 endonuclease activity is essential for AP site repair, Bcl2-mediated inhibition of APE1 endonuclease activity may suppress AP site repair. To test this possibility, Bcl2-overexpressing H1299 cells or vector-only control cells were treated with NNK for 60 min. The cells were then washed and incubated with normal cell culture medium for various times up to 72 h. AP sites of DNA lesions in genomic DNA were assessed using a DNA damage quantification (AP site counting) kit and analyzed using a microplate reader with a 650-nm filter as described under “Experimental Procedures.” The results reveal that NNK significantly enhances the AP sites of DNA lesions in both WT Bcl2-expressing and vector-only control cells within 60 min (Fig. 2C). After removal of NNK from the culture medium, AP sites of DNA lesions in vector-only control cells are significantly reduced within 24 h, indicating that most AP sites are repaired in vector-only control cells (Fig. 2C). By contrast, increased AP sites of DNA lesions are observed in Bcl2-overexpressing cells as compared with vector-only control cells after 24 h (Fig. 2C), suggesting that overexpression of Bcl2 potently inhibits the repair of NNK-induced AP sites of DNA lesions. Similar results were obtained from three independent clones expressing similar levels of exogenous Bcl2 (Fig. 2C). To test whether purified APE1 protein can restore the endonuclease activity in Bcl2 expressing extracts, increasing concentrations (i.e. 0.5–2.0 ng/ml) of purified APE1 protein were added to the nuclear extract(s) isolated from Bcl2-overexpressing H1299 cells in assay buffer. The results reveal that the addition of purified APE1 results in a dose-dependent increase in the production of the cleaved fragments (i.e. 14-mer) with a gradual decrease of the uncleaved 26-mer oligonucleotides (Fig. 2D), indicating that the addition of active APE1 is able to restore the endonuclease activity in Bcl2-expressing extracts. Exposure of Cells to the DNA-damaging Agent NNK Promotes Bcl2 Accumulation and Association with APE1 in Nucleus—Bcl2 is primarily localized in the outer mitochondrial membranes with minor expression in nuclear and endoplasmic reticulum membrane systems (17Hockenbery D. Nunez G. Milliman C. Schreiber R. Korsmeyer S. Nature. 1990; 348: 334-336Crossref PubMed Scopus (3544) Google Scholar, 18Zhu W. Cowie A. Wasfy G. Penn L.Z. Leber B. Andrews D. EMBO J. 1996; 15: 4130-4141Crossref PubMed Scopus (284) Google Scholar). Recent reports indicate that Bcl2 also resides in the nucleoplasm and functions within the nucleus (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Hoetelmans R. van Slooten H. Keijzer R. Erkeland S. van de Velde C. Dierendonck J. Cell Death Differ. 2000; 7: 384-392Crossref PubMed Scopus (88) Google Scholar, 20Schardle C. Li S. Re G. Fan W. Willinghamm M. J. Histochem. Cytochem. 1999; 47: 151-158Crossref PubMed Scopus (27) Google Scholar). We have previously demonstrated that the nuclear-localized Bcl2 has no antiapoptotic activity but is able to suppress DNA repair (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To test how Bcl2 regulates APE1 following DNA damage, H460 cells expressing high levels of endogenous Bcl2 and APE1 were exposed to NNK (5 μm) for 60 min. Subcellular distribution of Bcl2 was then examined by immunofluorescent staining. Consistent with our previous findings (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), the majority of Bcl2 is localized in cytoplasm, and only a small proportion is located in the nucleus in untreated cells. Intriguingly, Bcl2 is accumulated in nucleus after exposure of cells to NNK for 60 min (Fig. 3A). To further confirm this, subcellular fractionation was carried out to isolate mitochondrial and nuclear fractions. The results reveal that nuclear Bcl2 expression is enhanced within 60 min (Fig. 3B). After 3 h, the increased levels of nuclear Bcl2 are gradually reduced to the same level as the no treatment control (i.e. after the 6-h time point; Fig. 3B), indicating that NNK-enhanced nuclear Bcl2 expression occurs in a time-dependent manner. By contrast, the levels of mitochondrial Bcl2 show no significant change in the time course experiment (Fig. 3B, lower panel). Thus, increased nuclear Bcl2 may not result from a movement from mitochondria into nucleus. This effect on Bcl2 following exposure of cells to NNK may occur through a transcriptional or other unknown mechanism(s). Further study is required to demonstrate this possibility. To test for a direct interaction between Bcl2 and APE1 in nucleus, a co-immunoprecipitation was performed using isolated nuclear extract and an agarose-conjugated APE1 antibody. The results reveal that NNK-induced DNA damage promotes Bcl2 to associate with APE1 in a dose-dependent manner (Fig. 3C, upper panel). Thus, Bcl2 suppression of APE1 activity and AP site repair may occur in a novel mechanism by a direct interaction with APE1. Bcl2 Directly Interacts with APE1 via Its BH Domains, and Deletion of Any of the BH Domains from Bcl2 Results in Loss of the Ability of Bcl2 to Suppress APE1 Endonuclease Activity and AP Site Repair—Bcl2 family members share homology in the BH domains including BH1, BH2, BH3, and BH4 (21Kelekar A. Thompson C. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). To assess whether Bcl2 directly binds to APE1 via its BH domains, purified recombinant APE1 protein (10 ng) was incubated with purified recombinant WT, ΔBH1, ΔBH2, ΔBH3, or ΔBH4 Bcl2 deletion mutants (10 ng each) in 1% CHAPS lysis buffer at 4 °C for 2 h. The APE1-associated Bcl2 was co-immunoprecipitated with an agarose-conjugated APE1 antibody. The results demonstrate that APE1 is able to associate with WT but not with any of the ΔBH1, ΔBH2, ΔBH3, and ΔBH4 Bcl2 mutants (Fig. 4B, lower panel), indicating that all BH domains are essential for Bcl2 to interact with APE1. Because WT Bcl2 could not be immunoprecipitated by the APE1 antibody in the absence of APE1 (Fig. 4B, lower panel, lane 1 versus lane 2), this suggests that the binding of Bcl2 to APE1 is specific in this assay. To test whether Bcl2 protein directly affects APE1 endonuclease activity in vitro, a 32P-labeled, AP site mimetic, double-stranded F oligonucleotide was incubated with purified, active APE1 (1 ng/ml) in the absence or presence of purified recombinant WT or each of the BH deletion Bcl2 mutant proteins (1 ng/ml each) in the assay buffer as described under “Experimental Procedures.” The results reveal that purified APE1 efficiently cleaves the 26-mer oligonucleotides into a 14-mer fragment (Fig. 4C, lane 2). The addition of purified WT Bcl2 protein prevents the 26-mer oligonucleotide from cleavage induced by APE1 (Fig. 4C, lane 3), suggesting that Bcl2 can directly inhibit APE1 endonuclease activity in vitro. Intriguingly, all of the BH deletion Bcl2 mutant proteins fail to suppress APE1-induced cleavage of 26-mer oligonucleotide (Fig. 4C, lanes 4–7), indicating that deletion of any of the BH domains abrogates the capacity of Bcl2 to inhibit APE1 endonuclease activity. To functionally test this in vivo, APE1 endonuclease activity and AP sites of DNA lesions were compared in cells expressing WT or each of the BH deletion mutants. The results indicate that higher levels of APE1 endonuclease activity and accelerated AP site repair were observed in cells expressing each of the BH deletion mutants as compared with WT Bcl2-expressing cells (Fig. 4, D–F). This supports the notion that the binding of Bcl2 to APE1 via its BH domains may be required for the effect of Bcl2 on APE1 activity and AP site repair. Overexpression of Bcl2 in Cells Inhibits Formation of the APE1·XRCC1 Complex, and Purified Bcl2 Directly Disrupts the APE1·XRCC1 Interaction in Association with a Dose-dependent Inhibition of APE1 Endonuclease Activity in Vitro—It has been reported that a physical interaction between APE1 and XRCC1 significantly enhances APE1 endonuclease activity (22Vidal A. Boiteux S. Hickson I. Radicella J. EMBO J. 2001; 20: 6530-6539Crossref PubMed Scopus (403) Google Scholar). Because Bcl2 not only directly interacts with APE1 but also suppresses APE1 endonuclease activity in association with inhibition of AP site repair (Figs. 2, 3, 4), Bcl2 may affect the functional interaction between APE1 and XRCC1 via binding to APE1. To test this, association of APE1·XRCC1 was compared in Bcl2-overexpressing H1299 cells and vector-only control cells in the absence and presence of NNK. The results reveal that decreased levels of the APE1·XRCC1 complex were observed in the Bcl2-overexpressing cells as compared with vector control cells with or without NNK treatment, suggesting that expression of Bcl2 inhibits formation of the APE1·XRCC1 complex in cells (Fig. 5A). To further test whether Bcl2 can directly dissociate the APE1·XRCC1 complex in vitro, the APE1·XRCC1 complex was co-immunoprecipitated from H1299 parental cells expressing undetectable levels of endogenous Bcl2 using an APE1 antibody. The immune complex was incubated with increasing concentrations of purified, recombinant Bcl2 at 4 °C for up to 2 h, and proteins released from the complex were identified in the supernatant following centrifugation at 14,000 × g for 5 min. Surprisingly, Bcl2 directly disrupts the APE1·XRCC1 complex in vitro because the addition of purified Bcl2 results in decreased levels of bound XRCC1 on beads and increased levels of nonbound XRCC1 in the supernatant (Fig. 5B). Functionally, the addition of increasing concentrations (0.1–5 ng/ml) of purified, recombinant Bcl2 protein to the nuclear extract(s) isolated from H1299 parental cells results in a dose-dependent reduction in the production of the cleaved fragments (i.e. 14-mer) with a gradual increase of the uncleaved 26-mer oligonucleotides (Fig. 5C). Thus, in addition to a direct binding to APE1, Bcl2-mediated attenuation of APE1 endonuclease activity may also occur, at least in part, through disrupting a functional APE1·XRCC1 association. Specific Knockdown of Bcl2 Expression by RNA Interference Enhance APE1 Endonuclease Activity and Promotes AP Site Repair—To test a physiological role for Bcl2 in regulating APE1 activity and the repair of AP sites, a gene silencing approach was employed to specifically deplete the endogenous Bcl2 from H460 cells that express high levels of endogenous Bcl2. Recent studies have demonstrated that transfection of cells with siRNA concentrations greater than 100 nm frequently produces nonspecific off-target effects, and a concentration of 20–100 nm only occasionally produces off-target effects (23Cullen B. Nat. Methods. 2006; 3: 677-681Crossref PubMed Scopus (153) Google Scholar). However, siRNA concentrations of 10–20 nm generally do not exert nonspecific effects (24Semizarov D. Frost L. Sarthy A. Kroeger P. Halbert D. Fesik S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6347-6352Crossref PubMed Scopus (444) Google Scholar). To minimize nonspecific effects, we chose 10 nm of Bcl2 siRNA in the RNA interference experiment. The results reveal that transfection of Bcl2 siRNA significantly reduces the expression level of endogenous Bcl2 by more than 95% in H460 cells (Fig. 6A). This effect of siRNA on Bcl2 expression is highly specific because the control siRNA has no effect (Fig. 6A). Importantly, depletion of Bcl2 from H460 cells expressing high levels of endogenous Bcl2 up-regulates APE1 endonuclease activity (i.e. increased amount of the cleaved, 14-mer products) in association with accelerated AP site repair (Fig. 6, B and C). These findings provide strong evidence that physiologically expressed Bcl2 in cells is able to suppress AP site repair through a mechanism involving the inhibition of APE1 endonuclease activity. One of the most prevalent lesions in DNA is the AP site, which is the product of DNA glycosylases and the first DNA intermediate in the process of BER. If left unrepaired, AP sites are potentially genotoxic and/or mutagenic (25Loeb L. Cell. 1985; 40: 483-484Abstract Full Text PDF PubMed Scopus (246) Google Scholar). APE1, the second enzyme in BER pathway, initiates the repair of AP sites (22Vidal A. Boiteux S. Hickson I. Radicella J. EMBO J. 2001; 20: 6530-6539Crossref PubMed Scopus (403) Google Scholar). Growing evidence indicates that Bcl2 can suppress the repair of various types of DNA damage, including AP sites of DNA lesions, in association with increased genetic instability (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 10Kuo M. Shiah S. Wang C. Chuang S. Mol. Pharmacol. 1999; 55: 894-901PubMed Google Scholar, 11Liu Y. Naumovski L. Hanawalt P. Cancer Res. 1997; 57: 1650-1653PubMed Google Scholar, 12Saintigny Y. Dumay A. Lambert S. Lopez B. EMBO J. 2001; 20: 2596-2607Crossref PubMed Scopus (82) Google Scholar, 13Youn C.K. Cho H. Kim S. Kim H. Kim M. Chang I. Lee J. Chung M. Hahm K. You H. Nat. Cell Biol. 2005; 7: 137-147Crossref PubMed Scopus (67) Google Scholar, 26Chebonnel-Lasserre C. Gauny S. Kronenberg A. Oncogene. 1996; 13: 1489-1497PubMed Google Scholar). However, the molecular mechanism(s) by which Bcl2 regulates AP site repair remains elusive. Here we found that overexpression of Bcl2 suppresses APE1 endonuclease activity with decreased AP site repair (Fig. 2). Conversely, depletion of endogenous Bcl2 by RNA interference from H460 cells expressing high levels of endogenous Bcl2 enhances APE1 endonuclease activity in association with accelerated AP site repair (Fig. 6). These findings suggest that the inhibitory effect of Bcl2 on AP site repair may occur, at least in part, through down-regulating APE1 endonuclease activity. Bcl2 is mainly localized in mitochondrial membranes to maintain the mitochondrial integrity. Mounting evidence indicates that Bcl2 has also been found in the nucleoplasm and functions within the nucleus (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 19Hoetelmans R. van Slooten H. Keijzer R. Erkeland S. van de Velde C. Dierendonck J. Cell Death Differ. 2000; 7: 384-392Crossref PubMed Scopus (88) Google Scholar, 20Schardle C. Li S. Re G. Fan W. Willinghamm M. J. Histochem. Cytochem. 1999; 47: 151-158Crossref PubMed Scopus (27) Google Scholar). Intriguingly, the nuclear Bcl2 does not have antiapoptotic function but still has ability to regulate DNA repair (4Jin Z. May W.S. Gao F. Flagg T. Deng X. J. Biol. Chem. 2006; 281: 14446-14456Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). APE1 is mainly localized in the nucleus, but cytoplasmic APE1 can also be observed in some cell types (3Tell G. Damante G. Caldwell D. Kelley M. Antioxid. Redox Signal. 2005; 7: 367-384Crossref PubMed Scopus (327) Google Scholar). Here we found that NNK-induced DNA damage signal can stimulate Bcl2 accumulation in nucleus, which subsequently interacts with APE1 (Fig. 3). This may be a potential mechanism by which Bcl2 down-regulates APE1 activity with suppression of AP site DNA repair. Bcl2 family members share homology in regions designated the BH domains BH1, BH2, BH3, and BH4 (21Kelekar A. Thompson C. Trends Cell Biol. 1998; 8: 324-330Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar). Structure-function studies with Bcl2 deletion mutants reveal that APE1 directly interacts with Bcl2 and that all of the BH domains in Bcl2 are required for this interaction (Fig. 4, A and B). Because the BH1, BH2, and BH3 domains form the surface binding pocket of Bcl2 (27Castelli M. Reiners J. Kessel D. Cell Death Differ. 2004; 11: 906-914Crossref PubMed Scopus (24) Google Scholar), our findings suggest that in addition to the BH4 domain, the integrity of the surface binding pocket of Bcl2 is also important for Bcl2 to associate with APE1. Functionally, deletion of any of the BH domains abolishes the capacity of Bcl2 to suppress APE1 endonuclease activity as well as AP site repair (Fig. 4), indicating that the physical Bcl2-APE1 binding is required for the effects of Bcl2 on APE1 and AP site repair. XRCC1 acting as both a scaffold and a modulator of the different activities involved in BER provides a link between the incision and sealing steps of the AP site repair process. Intriguingly, XRCC1 not only physically interacts with APE1 but also potently stimulates its enzymatic activity (22Vidal A. Boiteux S. Hickson I. Radicella J. EMBO J. 2001; 20: 6530-6539Crossref PubMed Scopus (403) Google Scholar). Because overexpression of Bcl2 in cells or the addition of purified Bcl2 to the APE1·XRCC1 complex directly disrupts APE1·XRCC1 association (Fig. 5), this supports the notion that the inhibitory effect of Bcl2 on APE1 function may result from its physical binding to APE1 and/or subsequent disruption of the APE1·XRCC1 complex. In summary, our findings have uncovered a novel molecular mechanism by which Bcl2 suppresses AP site repair through inhibition of APE1 endonuclease activity. NNK-induced AP sites of DNA lesions facilitate Bcl2 accumulation in nucleus and interaction with APE1 via its BH domains, leading to decreased APE1 activity and attenuation of AP site repair. Because AP sites are mutagenic, Bcl2-mediated suppression of AP site repair may facilitate mutagenesis, genetic instability, and/or cancer development.
AP endonuclease
XRCC1
PARP1
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