Multifunctional human apurinic/apyrimidinic endonuclease 1: the role of additional functions

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is multifunctional enzyme. APEI is involved in the DNA base excision repair process (BER). APE1 participates in BER by cleaving the DNA adjacent to the 5' side of an AP site to produce a hydroxyl group at the 3' terminus of an unmodified nucleotide upstream of the nick and a 5' deoxyribose phosphate moiety downstream. In addition to its AP-endonucleolytic function, APE1 possesses 3' phosphodiesterase, 3'-5' exonuclease and 3' phosphatase activities. Independently of being characterized as DNA repair protein, APE1 was identified as redox-factor (Ref-1). Our own and literature data on the role of APE1 additional functions in cell metabolism and on interactions of APE1 with DNA and other proteins that participate in BER are analyzed in this review.     

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G^o) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar–phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5′ to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9–Rad1–Hus1 (9–1–1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein–DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.     

AP endonuclease 1 as a key enzyme in repair of apurinic/apyrimidinic sites

Human apurinic/apyrimidinic endonuclease 1 (APE1) is one of the key participants in the DNA base excision repair system. APE1 hydrolyzes DNA adjacent to the 5′-end of an apurinic/apyrimidinic (AP) site to produce a nick with a 3′-hydroxyl group and a 5′-deoxyribose phosphate moiety. APE1 exhibits 3′-phosphodiesterase, 3′-5′-exonuclease, and 3-phosphatase activities. APE1 was also identified as a redox factor (Ref-1). In this review, data on the role of APE1 in the DNA repair process and in other metabolic processes occurring in cells are analyzed as well as the interaction of this enzyme with DNA and other proteins participating in the repair system.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

Apurinic/apyrimidinic endonuclease (APE/REF-1) haploinsufficient mice display tissue-specific differences in DNA polymerase beta-dependent base excision repair

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 beta 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 beta-dependent BER response to cellular stress.     

Histones participate in base excision repair of 8-oxodGuo by transiently cross-linking with active repair intermediates in nucleosome core particles

8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) is a biomarker of oxidative DNA damage and can be repaired by hOGG1 and APE1 via the base excision repair (BER) pathway. In this work, we studied coordinated BER of 8-oxodGuo by hOGG1 and APE1 in nucleosome core particles and found that histones transiently formed DNA-protein cross-links (DPCs) with active repair intermediates such as 3′-phospho-α,β-unsaturated aldehyde (PUA) and 5′-deoxyribosephosphate (dRP). The effects of histone participation could be beneficial or deleterious to the BER process, depending on the circumstances. In the absence of APE1, histones enhanced the AP lyase activity of hOGG1 by cross-linking with 3′-PUA. However, the formed histone-PUA DPCs hampered the subsequent repair process. In the presence of APE1, both the AP lyase activity of hOGG1 and the formation of histone-PUA DPCs were suppressed. In this case, histones could catalyse removal of the 5′-dRP by transiently cross-linking with the active intermediate. That is, histones promoted the repair by acting as 5′-dRP lyases. Our findings demonstrate that histones participate in multiple steps of 8-oxodGuo repair in nucleosome core particles, highlighting the diverse roles that histones may play during DNA repair in eukaryotic cells.     

Y-box-binding protein 1 stimulates abasic site cleavage

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions. The first step in the AP site repair involves the magnesium-dependent enzyme AP endonuclease 1 (APE1) that catalyzes hydrolytic cleavage of the DNA phosphodiester bond at the 5′ side of the AP site, thereby generating a single-strand DNA break flanked by the 3′-OH and 5′-deoxyribose phosphate (dRP) groups. Increased APE1 activity in cancer cells might correlate with tumor chemoresistance to DNA-damaging treatment. It has been previously shown that the multifunctional oncoprotein Y-box-binding protein 1 (YB-1) interacts with APE1 and inhibits APE1-catalyzed hydrolysis of AP sites in single-stranded DNAs. In this work, we demonstrated that YB-1 stabilizes the APE1 complex with double-stranded DNAs containing the AP sites and stimulates cleavage of these AP sites at low magnesium concentrations.     

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

AP endonuclease (APE), with dual activities as an endonuclease and a 3' exonuclease, is a 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 spontaneously or after base excision during BER. It also removes the 3' blocking groups in DNA generated directly by ROS or after AP lyase reaction. In contrast to E. coli and lower eukaryotes which express two distinct APEs of Xth and Nfo types, mammalian genomes encode only one APE, APE1, which is of the Xth type. However, while the APEs together are dispensable in 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.     

XRCC1 interactions with base excision repair DNA intermediates

Abasic (AP) sites in DNA arise either spontaneously, or through glycosylase-catalyzed excision of damaged bases. Their removal by the base excision repair (BER) pathway avoids their mutagenic and cytotoxic consequences. XRCC1 coordinates and facilitates single-strand break (SSB) repair and BER in mammalian cells. We report that XRCC1, through its NTD and BRCT1 domains, has affinity for several DNA intermediates in BER. As shown by its capacity to form a covalent complex via Schiff base, XRCC1 binds AP sites. APE1 suppresses binding of XRCC1 to unincised AP sites however, affinity was higher when the DNA carried an AP-lyase- or APE1-incised AP site. The AP site binding capacity of XRCC1 is enhanced by the presence of strand interruptions in the opposite strand. Binding of XRCC1 to BER DNA intermediates could play an important role to warrant the accurate repair of damaged bases, AP sites or SSBs, in particular in the context of clustered DNA damage.     

AP endonuclease 1 coordinates flap endonuclease 1 and DNA ligase I activity in long patch base excision repair

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.     

Quantitative parameters of the 3′–5′ exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 with nicked DNA containing dYMP or a modified dCMP analogue

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, that incises DNA 5′ to the AP-site, it possesses other weak enzymatic activities. One of them is 3′–5′ exonuclease activity, which is most effectively exhibited for DNA duplexes containing modified or mismatched nucleotides at the 3′-end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase β through the base excision repair process. We determined the quantitative parameters of the 3′–5′ exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3′ terminus of single-strand DNA and of photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Isolation of a small molecule inhibitor of DNA base excision repair

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.     

Identification and characterization of mitochondrial abasic (AP)-endonuclease in mammalian cells

Abasic (AP)-endonuclease (APE) is responsible for repair of AP sites, and single-strand DNA breaks with 3′ blocking groups that are generated either spontaneously or during repair of damaged or abnormal bases via the DNA base excision repair (BER) pathway in both nucleus and mitochondria. Mammalian cells express only one nuclear APE, 36 kDa APE1, which is essential for survival. Mammalian mitochondrial (mt) BER enzymes other than mtAPE have been characterized. In order to identify and characterize mtAPE, we purified the APE activity from beef liver mitochondria to near homogeneity, and showed that the mtAPE which has 3-fold higher specific activity relative to APE1 is derived from the latter with deletion of 33 N-terminal residues which contain the nuclear localization signal. The mtAPE-sized product could be generated by incubating ³⁵S-labeled APE1 with crude mitochondrial extract, but not with cytosolic or nuclear extract, suggesting that cleavage of APE1 by a specific mitochondria-associated N-terminal peptidase is a prerequisite for mitochondrial import. The low abundance of mtAPE, particularly in cultured cells might be the reason for its earlier lack of detection by western analysis.     

AP endonuclease 1 prevents trinucleotide repeat expansion via a novel mechanism during base excision repair

Base excision repair (BER) of an oxidized base within a trinucleotide repeat (TNR) tract can lead to TNR expansions that are associated with over 40 human neurodegenerative diseases. This occurs as a result of DNA secondary structures such as hairpins formed during repair. We have previously shown that BER in a TNR hairpin loop can lead to removal of the hairpin, attenuating or preventing TNR expansions. Here, we further provide the first evidence that AP endonuclease 1 (APE1) prevented TNR expansions via its 3′-5′ exonuclease activity and stimulatory effect on DNA ligation during BER in a hairpin loop. Coordinating with flap endonuclease 1, the APE1 3′-5′ exonuclease activity cleaves the annealed upstream 3′-flap of a double-flap intermediate resulting from 5′-incision of an abasic site in the hairpin loop. Furthermore, APE1 stimulated DNA ligase I to resolve a long double-flap intermediate, thereby promoting hairpin removal and preventing TNR expansions.     

AP endonuclease 1 prevents the extension of a T/G mismatch by DNA polymerase β to prevent mutations in CpGs during base excision repair

Dynamics of DNA methylation and demethylation at CpG clusters are involved in gene regulation. CpG clusters have been identified as hot spots of mutagenesis because of their susceptibility to oxidative DNA damage. Damaged Cs and Gs at CpGs can disrupt a normal DNA methylation pattern through modulation of DNA methylation and demethylation, leading to mutations and deregulation of gene expression. DNA base excision repair (BER) plays a dual role of repairing oxidative DNA damage and mediating an active DNA demethylation pathway on CpG clusters through removal of a T/G mismatch resulting from deamination of a 5mC adjacent to a guanine that can be simultaneously damaged by oxidative stress. However, it remains unknown how BER processes clustered lesions in CpGs and what are the consequences from the repair of these lesions. In this study, we examined BER of an abasic lesion next to a DNA demethylation intermediate, the T/G mismatch in a CpG dinucleotide, and its effect on the integrity of CpGs. Surprisingly, we found that the abasic lesion completely abolished the activity of thymine DNA glycosylase (TDG) for removing the mismatched T. However, we found that APE1 could still efficiently incise the abasic lesion leaving a 3-terminus mismatched T, which was subsequently extended by pol β. This in turn resulted in a C to T transition mutation. Interestingly, we also found that APE1 3′–5′ exonuclease activity efficiently removed the mismatched T, thereby preventing pol β extension of the mismatched nucleotide and the resulting mutation. Our results demonstrate a crucial role of APE1 3′–5′ exonuclease activity in combating mutations in CpG clusters caused by an intermediate of DNA demethylation during BER.     

Study of interaction of XRCC1 with DNA and proteins of base excision repair by photoaffinity labeling technique

The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in base excision repair (BER) interacting with and modulating activity of key BER proteins. To estimate the influence of XRCC1 on interactions of BER proteins poly(ADP-ribose) polymerase 1 (PARP1), apurinic/apyrimidinic endonuclease 1 (APE1), flap endonuclease 1 (FEN1), and DNA polymerase beta (Pol beta) with DNA intermediates, photoaffinity labeling using different photoreactive DNA was carried out in the presence or absence of XRCC1. XRCC1 competes with APE1, FEN1, and PARP1 for DNA binding, while Pol beta increases the efficiency of XRCC1 modification. To study the interactions of XRCC1 with DNA and proteins at the initial stages of BER, DNA duplexes containing a photoreactive group in the template strand opposite the damage were designed. DNA duplexes with 8-oxoguanine or dihydrothymine opposite the photoreactive group were recognized and cleaved by specific DNA glycosylases (OGG1 or NTH1, correspondingly), although the rate of oxidized base excision in the photoreactive structures was lower than in normal substrates. XRCC1 does not display any specificity in recognition of DNA duplexes with damaged bases compared to regular DNA. A photoreactive group opposite a synthetic apurinic/apyrimidinic (AP) site (3-hydroxy-2-hydroxymethyltetrahydrofuran) weakly influences the incision efficiency of AP site analog by APE1. In the absence of magnesium ions, i.e. when incision of AP sites cannot occur, APE1 and XRCC1 compete for DNA binding when present together. However, in the presence of magnesium ions the level of XRCC1 modification increased upon APE1 addition, since APE1 creates nicked DNA duplex, which interacts with XRCC1 more efficiently.     

CUT Domains Stimulate Pol β Enzymatic Activities to Accelerate Completion of Base Excision Repair

The full-length CUX1 protein isoform was previously shown to function as an auxiliary factor in base excision repair (BER). Specifically, CUT domains within CUX1 stimulate the enzymatic activities of the OGG1 DNA glycosylase and APE1 endonuclease. Moreover, ectopic expression of CUX1 or CUT domains increased the resistance of cancer cells to treatments that cause oxidative DNA damage and mono-alkylation of bases. Stimulation of OGG1 AP/lyase and APE1 endonuclease activities, however, cannot explain how CUT domains confer resistance to these treatments since these enzymes produce DNA single-strand breaks that are highly toxic to cells. In the present study, we show that CUT domains stimulate the polymerase and deoxyribose phosphate (dRP)-lyase activities of DNA polymerase β to promote BER completion. In agreement with these results, CUX1 knockdown decreases BER completion in cell extracts and causes an increase in the number of abasic sites in genomic DNA following temozolomide treatment. We also show that CUT domains stimulate bypass of intrastrand G-crosslinks by Pol β in vitro, while the resistance of cancer cells to cisplatin treatment is reduced by CUX1 knockdown but restored by ectopic expression of CUT domains. Altogether our results establish CUX1 as an important auxiliary factor that stimulates multiple steps of base excision repair, from the recognition and removal of altered bases to the addition of new nucleotides and removal of 5′-deoxyribose phosphate required for ligation and BER completion. These findings provide a mechanistic explanation for the observed correlation between CUX1 expression and the resistance of cancer cells to genotoxic treatments.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.