122 Kinetics of apurinic/apyrimidinic endonuclease 1 (APE1) on an authentic AP site or an AP site analog

Our genomic DNA is endlessly exposed to a wide variety of exogenous and endogenous DNA-damaging agents. One of the most abundant DNA lesions is an apurinic/apyrimidinic (AP) site, which in vivo, can form spontaneously or through various cellular pathways, including the repair activity of DNA glycosylase enzymes (Wilson & Barsky, 2001). Persistence of these AP sites is both highly mutagenic and cytotoxic to the cell (Loeb & Preston, 1986). AP endonuclease 1 (APE1), an Mg²⁺ dependent enzyme, is the major human endonuclease responsible for incising the DNA backbone at AP sites. Repair to canonical duplex DNA is then completed by DNA polymerase and DNA ligase. Recently, APE1, in conjunction with delivery of DNA-damaging agents, has become a target for chemotherapeutic research with the aim to inhibit APE1 activity (Fishel & Kelley, 2007). Therefore, an understanding of APE1 activity and its molecular mechanism is essential. In vitro, the authentic AP site is highly unstable and can undergo β-elimination, leading to a strand break (Strauss, Beard, Patterson & Wilson, 1997). Due to the fragility of the AP site, stable AP site analogs, such as the reduced AP site or tetrahydrofuran (THF) site, are typically used to study APE1 (Maher & Bloom, 2007; Strauss, Beard, Patterson & Wilson, 1997). In this work, we have performed the first comprehensive kinetic study of APE1 acting on the authentic AP site as well the reduced AP site and THF AP site analog. Transient-state kinetic experiments reveal that the strand incision chemistry step is fast, upwards of ～700?s^?1 for all substrates, making APE1 one of the fastest DNA repair enzymes. Steady-state kinetic experiments reveal for each substrate, a slow, post chemistry step limits the steady-state rate. The steady-state rate for APE1 acting on authentic AP and AP-Red substrates is highly dependent on Mg²⁺ concentration, while the steady-state rate for THF site was not dependent on Mg²⁺ concentration. This comprehensive kinetic analysis reveal differences and similarities in the way APE1 processes the authentic AP site compared to AP site analogs. Furthermore, these differences require consideration when choosing AP site analogs to study APE1.     

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.     

Interaction of nucleotide excision repair protein XPC—RAD23B with DNA containing benzo[a]pyrene-derived adduct and apurinic/apyrimidinic site within a cluster

The combined action of reactive metabolites of benzo[a]pyrene (B[a]P) and oxidative stress can lead to cluster-type DNA damage that includes both a bulky lesion and an apurinic/apyrimidinic (AP) site, which are repaired by the nucleotide and base excision repair mechanisms — NER and BER, respectively. Interaction of NER protein XPC—RAD23B providing primary damage recognition with DNA duplexes containing a B[a]P-derived residue linked to the exocyclic amino group of a guanine (BPDE-N²-dG) in the central position of one strand and AP site in different positions of the other strand was analyzed. It was found that XPC—RAD23B crosslinks to DNA containing (+)-trans-BPDE-N²-dG more effectively than to DNA containing cis-isomer, independently of the AP site position in the opposite strand; protein affinity to DNA containing one of the BPDE-N²-dG isomers depends on the AP site position in the opposite strand. The influence of XPC—RAD23B on hydrolysis of an AP site clustered with BPDE-N²-dG catalyzed by the apurinic/apyrimidinic endonuclease 1 (APE1) was examined. XPC—RAD23B was shown to stimulate the endonuclease and inhibit the 3′–5′ exonuclease activity of APE1. These data demonstrate the possibility of cooperation of two proteins belonging to different DNA repair systems in the repair of cluster-type DNA damage.     

APE1 Incision Activity at Abasic Sites in Tandem Repeat Sequences

Repetitive DNA sequences, such as those present in microsatellites and minisatellites, telomeres, and trinucleotide repeats (linked to fragile X syndrome, Huntington disease, etc.), account for nearly 30% of the human genome. These domains exhibit enhanced susceptibility to oxidative attack to yield base modifications, strand breaks, and abasic sites; have a propensity to adopt non-canonical DNA forms modulated by the positions of the lesions; and, when not properly processed, can contribute to genome instability that underlies aging and disease development. Knowledge on the repair efficiencies of DNA damage within such repetitive sequences is therefore crucial for understanding the impact of such domains on genomic integrity. In the present study, using strategically designed oligonucleotide substrates, we determined the ability of human apurinic/apyrimidinic endonuclease 1 (APE1) to cleave at apurinic/apyrimidinic (AP) sites in a collection of tandem DNA repeat landscapes involving telomeric and CAG/CTG repeat sequences. Our studies reveal the differential influence of domain sequence, conformation, and AP site location/relative positioning on the efficiency of APE1 binding and strand incision. Intriguingly, our data demonstrate that APE1 endonuclease efficiency correlates with the thermodynamic stability of the DNA substrate. We discuss how these results have both predictive and mechanistic consequences for understanding the success and failure of repair protein activity associated with such oxidatively sensitive, conformationally plastic/dynamic repetitive DNA domains.     

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.     

Chlamydial DNA polymerase I can bypass lesions in vitro

We found that DNA polymerase I from Chlamydiophila pneumoniae AR39 (CpDNApolI) presents DNA-dependent DNA polymerase activity, but has no detectable 3' exonuclease activity. CpDNApolI-dependent DNA synthesis was performed using DNA templates carrying different lesions. DNAs containing 2'-deoxyuridine (dU), 2'-deoxyinosine (dI) or 2'-deoxy-8-oxo-guanosine (8-oxo-dG) served as templates as effectively as unmodified DNAs for CpDNApolI. Furthermore, the CpDNApolI could bypass natural apurinic/apyrimidinic sites (AP sites), deoxyribose (dR), and synthetic AP site tetrahydrofuran (THF). CpDNApolI could incorporate any dNMPs opposite both of dR and THF with the preference to dAMP-residue. CpDNApolI preferentially extended primer with 3'-dAMP opposite dR during DNA synthesis, however all four primers with various 3'-end nucleosides (dA, dT, dC, and dG) opposite THF could be extended by CpDNApolI. Efficiently bypassing of AP sites by CpDNApolI was hypothetically attributed to lack of 3' exonuclease activity.     

3′–5′ exonuclease activity of human apurinic/apyrimidinic endonuclease 1 towards DNAs containing dNMP and their modified analogs at the 3′ end of single strand DNA break

Human DNA apurinic/apyrimidinic (AP-) endonuclease 1 (APE1) is involved in the base excision repair (BER) pathway. The enzyme hydrolyzes DNA from the 5 side of the AP site. In addition to endonuclease activity, APE1 also possesses other slight activities including 3 -5 exonuclease activity. The latter is preferentially exhibited towards mispaired (non-canonical) nucleotides, this being the reason why APE1 is considered as a proofreading enzyme correcting the misincorporations introduced by DNA polymerase beta. We have studied 3 -5 exonuclease activity of APE1 towards dCMP and dTMP residues and modified dCMP analogs with photoreactive groups at the 3 end of the nicked DNA. Photoreactive dNMP residues were incorporated at the 3 end of the lesion using DNA polymerase beta and photoreactive dNTPs. The dependence of exonuclease activity on the "canonicity" of the base pair formed by dNMP flanking the nick at the 3 end, on the nature of the group flanking the nick at the 5 end, and on the reaction conditions has been determined. Optimal reaction conditions for the 3 -5 exonuclease hydrolysis reaction catalyzed by APE1 in vitro have been established, and conditions when photoreactive residues are not removed by APE1 have been chosen. These reaction conditions are suitable for using photoreactive nicked DNAs bearing 3 -photoreactive dNMP residues for photoaffinity labeling of proteins in cellular/nuclear extracts and model APE1-containing systems. We recommend using FAPdCTP for photoaffinity modification in APE1-containing systems because the FAPdCMP residue is less prone to exonuclease degradation, in contrast to FABOdCTP, which is not recommended.     

Conserved Structural Chemistry for Incision Activity in Structurally Non-homologous Apurinic/Apyrimidinic Endonuclease APE1 and Endonuclease IV DNA Repair Enzymes

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5′ AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5′ AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5′ to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg²⁺ and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg²⁺. Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.     

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.     

Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3' terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes

Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3' phosphodiesterase and 3'-5' exonuclease activities. The 3'-5' exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase beta. Here, we examine the removal of matched and mismatched dNMP from the 3' terminus of the 3'-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3' terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3' terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3'-5' exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5' margin of the nick. The 3'-5' exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5' margin of the nick as well as for flapped and recessed DNAs.     

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5′-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5′ direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3′ direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3′ direction (AP₊₃-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP₊₃-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3′ → 5′ exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.     

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5′ adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3′- and 5′-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5′-THF and the 3′-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5′-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.     

Reduced Nuclease Activity of Apurinic/Apyrimidinic Endonuclease (APE1) Variants on Nucleosomes: IDENTIFICATION OF ACCESS RESIDUES*

Non-coding apurinic/apyrimidinic (AP) sites are generated at high frequency in genomic DNA via spontaneous hydrolytic, damage-induced or enzyme-mediated base release. AP endonuclease 1 (APE1) is the predominant mammalian enzyme responsible for initiating removal of mutagenic and cytotoxic abasic lesions as part of the base excision repair (BER) pathway. We have examined here the ability of wild-type (WT) and a collection of variant/mutant APE1 proteins to cleave at an AP site within a nucleosome core particle. Our studies indicate that, in comparison to the WT protein and other variant/mutant enzymes, the incision activity of the tumor-associated variant R237C and the rare population variant G241R are uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. This defect appears to stem from an abnormal interaction of R237C and G241R with abasic DNA substrates, but is not simply due to a DNA binding defect, as the site-specific APE1 mutant Y128A, which displays markedly reduced AP-DNA complex stability, did not exhibit a similar hypersensitivity to nucleosome structures. Notably, this incision defect of R237C and G241R was observed on a pre-assembled DNA glycosylase·AP-DNA complex as well. Our results suggest that the BER enzyme, APE1, has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms.     

Ku antigen interacts with abasic sites

One of the most abundant lesions in DNA is the abasic (AP) sites arising spontaneously or as an intermediate in base excision repair. Certain proteins participating in the processing of these lesions form a Schiff base with the deoxyribose of the AP site. This intermediate can be stabilized by NaBH(4) treatment. By this method, DNA duplexes with AP sites were used to trap proteins in cell extracts. In HeLa cell extract, along with a prevalent trap product with an apparent molecular mass of 95 kDa, less intensive low-molecular-weight products were observed. The major one was identified as the p80-subunit of Ku antigen (Ku). Ku antigen, a DNA binding component of DNA-dependent protein kinase (DNA-PK), participates in double-stranded break repair and is responsible for the resistance of cells to ionizing radiation. The specificity of Ku interaction with AP sites was proven by more efficient competition of DNA duplexes with an analogue of abasic site than non-AP DNA. Ku80 was cross-linked to AP DNAs with different efficiencies depending on the size and position of strand interruptions opposite to AP sites. Ku antigen as a part of DNA-PK was shown to inhibit AP site cleavage by apurinic/apyrimidinic endonuclease 1.     

Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site

Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.     

Multifunctional human apurinic/apyrimidinic endonuclease 1: Role of additional functions

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme involved in base excision repair (BER). APE1 cleaves DNA 5′ of an AP site to produce a single-strand break with 5′-OH and 3′-deoxyribose phosphate. In addition to its AP-endonucleolytic function, APE1 possesses 3′-phosphodiesterase, 3′–5′ exonuclease, and 3′-phosphatase activities. Independently of its function as a repair protein, APE1 was identified as a redox factor (Ref-1). The review summarizes the published and original data on the role of the additional functions of APE1 in DNA repair and apoptosis and regulation of the BER system via APE1 interaction with DNA and other repair proteins.     

Modulation of the processive abasic site lyase activity of a pyrimidine dimer glycosylase

The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.     

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

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg²⁺, while ssDNA AP site cleavage required Mg²⁺ (optimally at 0.5-2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3′-PO₄²⁻ group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.