Intracellular trafficking and regulation of mammalian AP-endonuclease 1 (APE1), an essential DNA repair protein

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Intracellular trafficking and regulation of mammalian AP-endonuclease 1 (APE1), an essential DNA repair protein Sankar Mitra a,∗ , Tadahide Izumi c , Istvan Boldogh b , Kishor K. Bhakat a , Ranajoy Chattopadhyay a , Bartosz Szczesny a a

Sealy Center for Molecular Science and Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, United States b Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX 77555, United States c Louisiana State University Health Sciences Center, New Orleans, LA 70112, United States

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Article history:

AP endonuclease (APE), with dual activities as an endonuclease and a 3 exonuclease, is a

Published on line 12 December 2006

central player in repair of oxidized and alkylated bases in the genome via the base excision repair (BER) pathway. APE acts as an endonuclease in repairing AP sites generated spon-

Keywords:

taneously or after base excision during BER. It also removes the 3 blocking groups in DNA

Nuclear export signal

generated directly by ROS or after AP lyase reaction. In contrast to E. coli and lower eukaryotes

Age-dependent repair activity

which express two distinct APEs of Xth and Nfo types, mammalian genomes encode only

Mitochondrial APE

one APE, APE1, which is of the Xth type. However, while the APEs together are dispensable in

Spontaneous AP sites

the bacteria and simple eukaryotes, APE1 is essential for mammalian cells. We have shown that apoptosis of mouse embryo fibroblasts triggered by APE1 inactivation can be prevented by ectopic expression of repair competent but not repair-defective APE1. The mitochondrial APE (mtAPE) is an N-terminal truncation product of APE1. A significant fraction of APE1 is cytosolic, and oxidative stress induces its nuclear and mitochondrial translocation. Such age-dependent increase in APE activity in the nucleus and mitochondria is consistent with the hypothesis that aging is associated with chronic oxidative stress. © 2006 Elsevier B.V. All rights reserved.

1.

Introduction

Endogenous damage continuously inflicted on the genomes of all organisms has several bases. Spontaneous chemical reactions occur in DNA which include depurination and cytosine deamination that generate AP sites (APS) and uracil (U), respectively [1]. Excision of U by uracil-DNA glycosylase then generates APS [2,3]. Repair of other endogenous genomic lesions, namely methyl and ethenobase adducts, by DNA

glycosylases, also generates APS [2,4]. The noninstructional APS prevent both replication and transcription, and hence are toxic [5,6]. They are also likely to be mutagenic because of their replication by lesion bypass DNA polymerases [6,7]. The number of APS generated has been estimated to be ∼104 /cell/day at 37 ◦ C and the steady-state level of APS as 104 –105 /cell [8–11]. In addition to the spontaneous DNA damage, oxidative damage constitutes the most pervasive endogenous damage occurring in the genomes of all aerobic organisms. Reactive

∗ Corresponding author at: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.136 Medical Research Building, Route 1079, Galveston, TX, United States. Tel.: +1 409 772 1788; fax: +1 409 747 8608. E-mail address: [email protected] (S. Mitra). 1568-7864/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2006.10.010

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oxygen species (ROS) are continuously generated as respiration by-products (up to 1–5% of consumed O2 ) in these organisms, initially as the O2 −• superoxide anion radical due to partial reduction of O2 . Although O2 −• is also generated by various oxidases, its major source are the mitochondria [12]. O2 −• is converted into H2 O2 , another ROS, by the ubiquitous superoxide dismutase (SOD). H2 O2 can then give rise to the OH• radical, the most reactive ROS, by reaction with O2 −• or via Fenton reaction. The NOX radical produced as a signaling molecule by NO synthase reacts with O2 −• to generate peroxynitrite which again could breakdown into OH• [13]. The singlet O2 is generated during photochemical oxidation of cellular components, e.g., riboflavin exposed to long wave length UV light [14]. In specialized cells such as activated neutrophils, HOCl, another ROS, is generated due to combined action of SOD and myeloperoxidase [15]. The impact of ROS on various cellular processes has yet to be completely delineated. Broadly, ROS triggers a multitude of signaling events initiated with oxidation of lipids and proteins. In spite of some recent contradictory reports [16], the aging syndrome in mammals is commonly believed to be complex manifestation of chronic oxidative stress [17]. There is significant evidence in support of the hypothesis that age-dependent decline in mitochondrial integrity and hence function is responsible for the chronic, endogenous oxidative stress. The mitochondria are in a unique situation in that the initial ROS damage to mitochondrial proteins and genomes leads to increased leakage of O2 −• from the respiratory complexes which in turn causes more damage to the organelle. Thus such ROS amplification is the result of a positive feedback loop starting with a low level of ROS damage to the mitochondria. ROS damages both mitochondrial and nuclear genomes; however, it is now clear that in oxidatively stressed cells, the mitochondrial genome is significantly more susceptible to oxidative damage than the nuclear genome [18].

bleomycin [9,21]. DNA single-strand break repair (SSBR) is considered as a subpathway of BER because it utilizes the same enzymes except for the DNA glycosylases. The first common enzyme in BER and SSBR in all organisms is APE [2,5,20].

3.

Central role of APE in BER/SSBR

All APEs have dual activities. As endonucleases these cleave the DNA strand 5 to the AP site, or as 3 exonucleases remove 3 blocking groups generated chemically or enzymatically, to provide the 3 OH terminus needed for subsequent repair synthesis [5,20]. As with most BER enzymes, the E. coli APEs were the first to be characterized. Xth (exonuclease III), discovered as a 3 exonuclease, was subsequently characterized as the major APE in E. coli [22,23]. Nfo (endonuclease IV) was later identified as the second APE in E. coli, with completely different structure and amino acid sequence [24]. Inactivation of the nfo gene increases sensitivity to bleomycin whereas xth mutation sensitizes E. coli to alkylating and oxidizing agents [24]. Interestingly, Xth and Nfo have similar substrates but Nfo is uniquely involved in repair of ␣-anomers of adenosine [25,26]. Additionally, Nfo does not have potent 3 exonuclease activity for undamaged DNA, although it does have the 3 end cleaning activity common to all APEs. Because of the fundamental difference in their structures, Nfo and Xth are considered as prototypes of the two families of APEs. Two APE’s belonging to Xth and Nfo families, respectively have been identified in eukaryotes including both fission and budding yeasts, and in C. elegans. These have been named Apn1 (Nfo homolog) or Apn2 [Xth homolog; 27,28,29,30]. These have not been thoroughly characterized.

4. Mammalian cells express a single APE, APE1 2. Oxidative damage is repaired via the DNA base excision repair (BER) pathway BER represents the simplest type of excision repair, and is active in both nucleus and mitochondria. Nearly all oxidized forms of DNA bases as well as methylated or inappropriate bases such as U are repaired via the BER pathway which is initiated with excision of the damaged base by a DNA glycosylase. All mammalian oxidized base-specific glycosylases, namely OGG1, NTH1, NEIL1 and NEIL2, have intrinsic AP lyase activity which causes cleavage of the resulting APS after base excision via ␤ or ␤␦ elimination [3,19,20]. The cleaved DNA strand in all cases contains 3 blocking phosphodeoxyribose (3 phospho ␣,␤-unsaturated aldehyde) or 3 phosphate which has to be removed prior to the filling of the resulting gap by a DNA polymerase. Furthermore, in all cases the 5 terminus contains phosphate which allows DNA ligase-mediated sealing of the nick after gap filling. Thus, removal of the 3 blocking group is essential in repair of oxidized bases. ROS directly reacts with the deoxyribose at Cl or C3 and thereby causes strand breaks with AP sites or oxidized AP sites most of which have blocked 3 termini. 3 phosphoglycolate generated at DNA single strand breaks is a common end product of ROS or ROS generators, e.g.,

Unlike bacteria and lower eukaryotes, the mammals express only one APE, named APE1, which belongs to the Xth family [5,31]. Recently, a second mammalian Xth-type APE candidate, (APE2) was cloned [32,33]; however, we could not detect APE activity in the recombinant 66 kD protein (Wiederhold, L., unpublished experiment). That human APE2 (unlike APE1) cannot complement APE-negative S. pombe mutants [30] supports our conclusion that APE1 is the sole APE in mammals. APE1 has dual activities like other APEs. However, APE1 s endonuclease activity is quite strong while its 3 exonuclease/phosphodiesterase activity is weak [5]. In fact, APE1 has barely detectable DNA 3 phosphatase activity, in contrast to robust DNA 3 phosphatase activity of E. coli Xth [5,34]. The 3 phosphate termini are directly generated at ROS-induced single-strand breaks, and also due to ␤␦ lyase activity of DNA glycosylases of the Fpg/Nei type, including mammalian NEIL1 and NEIL2 [35–37]. Unlike E. coli, mammalian cells express high levels of polynucleotide kinase (PNK) which has dual activities as a 5 nucleotide kinase and 3 phosphatase [38,39]. We have shown that human NEIL1-dependent repair utilizes PNK rather than APE1 [34]. We also raised the possibility that APE1 is dispensable for repair of AP sites in the presence of both

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NEILs and PNK [40]. Nevertheless, APE provides the major APS repair activity in BER in mammalian cells.

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Contrary to a common perception, many repair proteins (and other proteins with exclusively nuclear functions) are present both in the nucleus and cytosol. The repair proteins are transiently targeted to the nucleus and mitochondria in response to DNA damage signals [20]. Although oxidative stress increases APE1 s level and its nuclear accumulation [41], we did not examine the mechanism of its nuclear import before. Recently we confirmed an earlier prediction that APE1 s nuclear transport required its 20 N-terminal residues containing a nuclear localization sequence (NLS) in residues 2–8, including acetylation sites Lys6/Lys7 [42,43]. Fig. 1 demonstrates that unlike full-length APE 1-FLAG, which was predominantly nuclear (Fig. 1 A), nuclear localization of both N-terminal deletion mutants N13 and N16 APE1-FLAG are significantly perturbed with the presence of a much higher level in the cytoplasm relative to the nucleus (Fig. 1B, C). At the same time, we observed that deletion of the 7 N-terminal residues did not completely inhibit APE1 s nuclear translocation. Further, APE1 s nuclear accumulation after treatment with leptomycin B, a specific inhibitor of nuclear export, suggested that APE1 s N-terminal sequence contains a nuclear export signal (NES) as well [43]. Our results thus suggest that oxidative stress-induced nuclear accumulation of APE1 may be regulated more by inhibition of nuclear export than via enhanced import [43,44].

heteroplasmy [45,46]. The other key feature of the mitochondrial genome is its higher susceptibility to oxidative stress, as mentioned earlier. In addition to their proximity to the sites of ROS generation, it is likely that the mitochondrial genomes are more prone to oxidative damage because histones and other chromatin-associated proteins, present in nuclear genomes and acting as scavengers of oxygen radicals, are absent in the mitochondria. In any case, the issue of whether oxidative damage to the mitochondrial genome is repaired in vivo was settled several years ago [47–49]. Even before those studies, mitochondria-specific BER enzymes, in particular, DNA glycosylases, e.g., uracil-DNA glycosylase, was discovered [3]. Mitochondrial DNA polymerase ␥ (Pol␥) and DNA ligase III␣ (Lig III␣), which are involved in mitochondrial DNA replication, appear to be functional in mitochondrial BER as well [50,51]. It is interesting to note that the replicative DNA polymerase, Pol␥, is unique for the mitochondria, while the mitochondrial Ligase III␣ is a splice variant of the nuclear Lig III␣ [52]. Similarly, mitochondria-specific OGG1 and NTH1, the major DNA glycosylases specific for repair of oxidatively damaged bases in the nuclear genome, have been identified and characterized. These are splice variants of the corresponding nuclear enzymes [53,54]. The mitochondrial isozymes are generated primarily to eliminate the nuclear localization signal (NLS) of such proteins which is required for their targeting to the nucleus [55]. Mitochondrial targeting often requires an N-terminal mitochondrial targeting sequence (MTS) which is cleaved off in the mitochondrial matrix by specific peptidases [56]. However, some mitochondrial proteins constitutively lack the MTS, and appear to utilize an internal MTS which is not removed by proteolysis during their translocation to the mitochondrial matrix [57].

6. Repair of oxidative damage in mitochondrial genomes via BER pathway

7. Characterization of mitochondrial APE (mtAPE)

The mitochondrial genome constituting a few percent of the total DNA in mammalian cells has several unique features. Each cell could have hundreds of mitochondria with each containing 10–100 or so of small circular genomes (∼16 kb). The genomes could tolerate heterogeneity in sequence, named

It is interesting to note that that while most BER proteins needed to repair oxidized bases in the mitochondrial genomes have been characterized, the origin of mitochondrial APE (mtAPE) was still not clear. An earlier study suggested the presence of a 66–68 kDa, mt-specific APE polypeptide [58].

5. Nuclear localization and export signals in APE1

Fig. 1 – Analysis of APE1 s N-terminal sequence for nuclear localization using C-terminally fused FLAG. Full-length hAPE1-FLAG (A), N13 APE 1-FLAG (B), N16 APE 1-FLAG (C) were analyzed for their intracellular localization by anti-FLAG antibody conjugated to FITC (green). PI, propidium iodide staining; TM, transmission; SI, superimposition of FITC and PI fluorescence.

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Although APE2 was identified in the nucleus and mitochondria, as already mentioned, the recombinant protein shows no APE activity. Finally, the presence of APE1 in mitochondria was verified by several groups usually via immunohistochemical analysis; however the mtAPE was not purified and characterized [51,59]. Because of earlier contradictory results about the identity of mammalian mtAPE, we decided to characterize this enzyme, using the ab initio approach. We purified and characterized mtAPE from purified mitochondria of beef liver. Because the “purified” mitochondria are often contaminated with nascent enzymes synthesized in the endoplasmic reticulum (ER), which is intimately connected with mitochondrial membrane, and with cytosolic contaminants, it is possible that APE1 observed in some mitochondrial preparations in earlier studies were nuclear/cytosolic contaminants. Because of this, we routinely treat the intact mitochondria with trypsin which does not affect enzymes within the mitochondrial matrix [60]. Using this stringent criterion, we succeeded in purifying mtAPE based on activity, to a point where the major protein band corresponding to APE activity could be analyzed by MALDI-TOF and chemical sequencing. We established that mtAPE is a truncation product of APE1, with loss of 33 Nterminal residues [61]. We observed the presence of truncated APE1 proteins in the mitochondrial matrix of not only bovine liver but also in mouse liver and NIH 3T3 cell line [61]. We unexpectedly observed that the specific activity of mtAPE purified from beef liver was 2- to 3-fold higher than that of full-length APE1. Because the first 61 amino acid residues of APE1 are not required for its repair activity [62], we concluded that the N-terminal residues in wild type (WT) APE1 is indirectly involved in BER by enhancing coordination via product binding and interaction with downstream repair proteins, including Pol␤ and PCNA [63]. It is thus possible that, unlike nuclear APE1, mtAPE lacking the N-terminal domain is not involved in stable interaction with other proteins for repair.

8. Mitochondrial APE, intracellular distribution and aging The free radical theory of aging was first proposed by Harman who hypothesized that aging occurs due to free radicals, products of aerobic respirations, which cause cumulative oxidative damage to DNA, proteins and lipids [64]. Identification of the mitochondria as the major source of ROS is consistent with the mitochondrial theory of aging. Schriner et al. [65] showed that mitochondria-specific ectopic expression of catalase, expected to inactivate H2 O2 generated in situ, increases lifespan of transgenic mice by 5 months with corresponding decrease in cardiac pathology and cataract formation, and reduction in oxidative damage and deletions in mitochondrial genomes. This strongly supports the possibility that at least in highly oxygenated tissues, mitochondrial ROS is involved in the etiology of the aging syndrome. In this scenario, mitochondrial mutations accumulating with age contribute to increased production of ROS and susceptibility to various diseases [66,67]. We have previously shown that activation of APE1 is mediated by ROS [41]. An age-dependent decrease in stress-induced responses to oxidative insults in rats was

Table 1 – Age-dependent modulation of APE activity in mouse liver organelles (fmol/min/␮g protein)

Total extract Nuclear Mitochondria

4-mo

10-mo

20-mo

23.6 ± 1.2 38.0 ± 7.6 1.5 ± 0.7

19.6 ± 1.7 48.5 ± 5.9 9.6 ± 2.5

23.2 ± 2.8 72.0 ± 10.8 9.6 ± 2.1

observed with a significant increase in the APE1 protein level after oxygen exposure in 3-mo old rats, but not in 30-mo old rats. These results raise the possibility that the age-dependent decline in response to oxidative insults may result in deficiency in repair of oxidative DNA damage [68]. Cells exposed to a low dose of ROS significantly increased their resistance to cytotoxicity, due to enhanced repair of the oxidative DNA damage [69]. We have shown recently that there is no significant agedependent change in total APE1 protein and activity in the mouse liver. However, we did observe an age-dependent difference in intracellular distribution of the APE activity (Table 1). Two and 6-fold increases in specific activity of APE were found in the nucleus and mitochondria, respectively, of old livers compared to the livers of young animals [70]. This was associated with a decrease in the cytoplasmic APE activity [70]. Our results thus indicate accumulation of APE in the nucleus and mitochondria as a function of age due to redistribution of the enzyme from the cytosol. Furthermore, such age-dependent translocation occurs earlier in the mitochondria than in the nucleus. These results may explain lack of induction of APE1 in old rats after exposure to hyperoxia [68] because such activation and nuclear accumulation of APE1 has already occurred as a function of age. An age-dependent increase in the steady state level of ROS was documented earlier in several studies [71]. We and others have shown translocation of APE1 to the nucleus and mitochondria in response to exogenous oxidative stress [41,69,72]. We had proposed that increased accumulation of APE1 in the nucleus and mitochondria in old mouse hepatocytes is induced by higher endogenous oxidative stress in the old animals [70]. We could rationalize these observations by postulating that the altered distribution of this key repair protein is necessary to cope with the enhanced oxidative damage to nuclear and mitochondrial genomes in older animals. It was reported that mitochondrial APE activities were marginally lower in all tissues of mice subjected to caloric restriction, except in the brain, where 50% decrease in APE activity was observed [73]. It was further suggested that the capacity for mitochondrial DNA repair in these tissues is regulated either by the level of oxidative lesions in mtDNA or directly by ROS [73]. An increase in the level of AP sites in the genome as a function of age was shown earlier [74]. Interesting information was obtained from the analysis of APE1 expression and intracellular localization of APE1 in normal and neoplastic tissues. Normal breast tissues showed mostly nuclear localization of APE1, while in the more aggressive tumors, the predominant staining for APE1 was cytoplasmic [75,76]. These results paint a complex picture of intracellular distribution and trafficking of APE1 which is controlled by multiple parameters. Further studies are needed for elucidating the mechanism of subcellular targeting of APE1 and to test whether the dynamics of intracellular translocation is unique

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for APE1 because of its multiple functions. These studies could also provide critical insight regarding therapeutic approaches.

9.

Proteolytic processing of APE1

Granzyme A, the most abundant granzyme expressed in cytolytic T and natural killer cells, causes death of target cells via caspase-independent mechanisms. It recognizes a large chromatin modifying endoplasmic reticulum-associated SET complex. Cleavage of SET protein results in activation of a single strand-specific DNase (NM23-H1) and its translocation to the nucleus [77,78]. Granzyme A causes typical apoptotic features, e.g., membrane perturbation, chromatic condensation and loss of mitochondrial membrane potential [79]. Recently, one of the subunits of the SET complex was identified to be APE1. It was further postulated that Granzyme A cleaves APE1 at Lys31 and inactivates it. As a result, DNA repair is blocked which in turn triggers apoptosis [80]. However, the fact that 61 N-terminal amino acid residues of APE1 are not required for its endonuclease activity was not considered [62]. It was independently reported that N-terminal truncation of APE1 (AN34) is linked to DNA fragmentation during apoptosis AN34 APE1, generated due to cleavage by caspase 3; however, the full-length APE1 did not cleave undamaged chromatin DNA [81]. This result was supported by the observation that silencing of APE1 expression strongly suppresses apoptotic DNA fragmentation in the U87-MG cell line. The complex picture of enzymatic cleavage of APE1 in vivo warrants comprehensive analysis of APE1’s processing in various cell types.

10.

APE1 is essential in mammals

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nucleotide excision repair (NER) in E. coli, in the APE double mutant background was unsuccessful [87]. Given that APS could be repaired via the NER pathway to some extent [88], this result implies that E. coli may not survive without some APS repairing activity. S. cerevisiae lacking both APN1 and APN2, genes like E. coli, genes grow normally although the mutant is similarly hypersensitive to MMS and H2 O2 . Another striking similarity to E. coli was recently found by Guillet and Boiteux recently in that the yeast triple mutant lacking both APE and an NER protein, RAD1 or RAD10, could not grow beyond 10 divisions [89]. This suggests that a basal activity for APS repair is essential in all organisms. Because only one APE (APE1) has been identified in mammalian cells, it is possible that backup proteins with APE activity exist in these cells also, as is the case for some other BER enzymes. Such activity may not be detectable in wild type cells in the presence of APE1 s strong activity. We therefore deemed it important to test survival of APE1-negative cells.

12. APE1 is essential in mammalian fibroblasts We have established conditional APE1-null mouse primary embryonic fibroblast (MEF) lines from APE1 conditional null mouse mutants. We showed using nuclear microinjection that deletion of the “floxed” human APE1 transgene by Cre recombinase induces apoptosis in these MEFnull cells, which is prevented by ectopic expression of WT APE1 [90]. Apoptosis could be prevented by simultaneous expression of APE1 mutants lacking either the regulatory or DNA repair activity, but not both [90]. To further analyze the impact of APE1 inactivation in the conditional mutant cells in mass culture, we

APE1 was shown to be essential during early development of mouse embryos [82]. The APE1 homozygous null mice die just after blastocyst formation (3–5 days after fertilization). The lethality due APE1 null mutation was later confirmed by two other groups [83,84]. It was however still unclear which function(s) of APE1 are absolutely required during early embryogenesis. We should note that no mouse embryonic fibroblast cells (MEF) were ever isolated from the APE1 −/− embryos. This presents a striking contrast to the situation with other BER genes such as DNA polymerase ␤ (pol␤) and XRCC1 which were also shown to be essential for mouse embryo development. However, mutant MEFs lacking these proteins are viable and have contributed greatly to understanding their functions and role in BER [85,86]. It appeared possible that the inability to isolate APE1 −/− MEFs could be due to technical reasons.

11. Requirement of APE activity in other organisms Is APE activity absolutely required for all organisms? This is an interesting question which has not been addressed clearly even in prokaryotic cells. E. coli strains lacking both APE genes grow normally, although the cells show a high sensitivity to reactive oxygen species (e.g., H2 O2 ), and alkylating reagents such as methyl methanesulfonate which generates APS [24]. However, an attempt to delete the uvrA gene required for

Fig. 2 – Cre-mediated loss of APE1 and increase in DNA AP sites of MEFnull infected with Cre lentivirus. (A) Western analysis for APE1 depletion and Cre expression (40 ␮g extract). (B) AP sites in empty (Con) or Cre virus-infected cells.

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Fig. 3 – Apoptosis of MEFnull at 24 h after infection with empty lentivirus control (Con) or Cre virus. (A) Apoptotic cells after Cre expression (18 h), treated with Cre antibody and FITC-conjugated secondary antibody. (B) increase in the level of apoptotic cells after Cre expression (18 h) in MEFnull . Chlorambucil was used as a control inducer of apoptosis. (C) 2-D FACS analysis of Cre-expressing or chlorambucil-treated cells after staining with annexin V and 7AAD.

transduced these cells with Cre-expressing lentiviral vector developed by Xiao-Feng Qin [91]. Western analysis of infected cell extracts shows time-dependent increase in Cre expression with concomitant depletion of APE1 (Fig. 2A). In order to test our hypothesis that APS are continuously generated in the genome and repaired by APE1, we analyzed the amount of APS in APE1-depleted cells. We treated MEFnull with an aldehydereactive probe (ARP), at 48 h after Cre virus infection [10,92]. Fig. 2B shows significant increase in the level of APS. We confirmed that MEFnull cells underwent apoptosis in the absence of APE1, in mass culture. The majority (80–90%) of these cells showed characteristic morphology of apoptotic cells at 24 h after Cre virus infection (Fig. 3 A). This was further confirmed by 2-D FACS analysis in cells stained with annexin V and 7-amino actinomycin D (7-ADD) which stains necrotic cells (Fig. 3B, C). It is thus evident that APE1 inactivation induces necrosis in addition to apoptosis. Taken together, our results show that APE1 is essential in somatic cells, and that its repair and regulatory functions are both essential and separable. Thus some or all endogenous APS (and possibly their oxidation products) in the genome trigger apoptosis and necrosis. Fung and Demple [93] also showed essentiality of APE1 in several tumor cell lines in which APE1 was downregulated by siRNA. They demonstrated that apoptosis due to APE1 deficiency could be prevented by

ectopic APN1 of S. cerevisiae. Several earlier studies had provided evidence for apoptosis induced by APE1 downregulation in neuronal and other lines [reviewed in 94]. In any case, these results suggest that unlike in E. coli or yeast, where APS could be repaired via the NER pathway in the absence of APEs, a similar back-up system for APS repair is absent in mammalian cells.

13.

Concluding remarks

Based on our results of apoptosis of MEF after APE1 inactivation, it is highly likely that APE activity is essential for all somatic cells [90]. Although we have not purposely discussed such BER-unrelated regulatory activities of APE1 in this review, we showed that APE1 s regulatory activity is also essential for MEFs. There are still many unanswered questions regarding the repair activity of APE1. The signaling for APE1 s nuclear or mitochondrial translocation has not been elucidated. Our preliminary results indicate that, unlike some other BER proteins, APE1 has a rather short half-life. Thus there appears to be a complex regulatory circuit for APE1 which couples its de novo synthesis and intracellular translocation in response to oxidative stress. Furthermore, frequent overexpression of APE1 in tumor cells strongly suggests its role in preventing

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tumor cell apoptosis possibly by repairing some endogenous damage which are yet to be identified. It is thus evident that APE1 will continue to be a topic of vigorous investigation for many years to come.

Acknowledgments This review article is dedicated to the memory of Dr. Erling Seeberg whose untimely death has dealt a major blow to the field of DNA repair. Although Dr. Seeberg was interested in nucleotide excision repair early in his career, his subsequent seminal studies in elucidating the initial steps in base excision repair, and repair via direct damage reversal, significantly contributed to our present understanding of oxidative and alkylation damage repair in mammalian genomes. The original research described in this review was supported by USPHS R01 ES08457, R01 CA53791 (SM), P30 ES06676, P01 AG-21803 (IB, SM), R01 CA98664 (TI). We express our sincere gratitude to Xiao-Feng Qin (M.D. Anderson Cancer Center, Houston, TX) for providing the lentiviral vector and critical guidance for generating recombinant lentiviruses. We also thank Wanda Smith for expert secretarial assistance and UTMB Cell Sorting Facility for FACS analysis.

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