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.     

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.     

The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease,ARP is involved in the plant nucleotide incision repair pathway

Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3' → 5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (k_cat/K_M = 134 and 7.3 μM⁻¹·min⁻¹, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3' → 5' exonuclease activities (k_cat/K_M = 314 and 34 μM⁻¹·min⁻¹, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp^–/– mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H₂O₂, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.     

Interactions of human ribosomal protein S3 with intact and damaged DNA

Human S3 (hS3) is a structural component of the ribosome and, in addition to its role in translation, possesses apurinic/apyrimidinic (AP) lyase activity, characteristic of DNA repair enzymes. Recombinant hS3 was isolated from inclusion bodies, refolded under different conditions, and tested for the ability to bind and cleave oligodeoxyribonucleotide substrates with various lesions abundant in genomic DNA: AP sites, uracil, 8-oxoguanine, 8-oxoadenine, 5,6-dihydrouracil, and hypoxanthine. It was found that hS3 is capable of cleaving AP sites via the β-elimination mechanism, producing a Schiff base covalent intermediate, but cannot cleave substrates with the other lesions. Refolding in the presence of Fe²⁺ and S^2? did not increase hS3 activity, suggesting the absence of an iron-sulfur cluster. The binding of hS3 with DNA ligands containing oxidized or deaminated bases was less efficient than with intact DNA. It was assumed that the catalytic activity of hS3 towards AP sites is most likely unimportant for global DNA repair in vivo, but is possibly involved in repairing DNA sites in certain genome regions.     

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3′-end-blocking modifications, possesses DNA and RNA 3′-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3′-end of DNA have been used to prevent 3′-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3′-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3′-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3′-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site 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.     

Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III

Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease III (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its AP endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair.     

Distinct roles of Ape1 protein in the repair of DNA damage induced by ionizing radiation or bleomycin

Ionizing radiation (IR) and bleomycin (BLM) are used to treat various types of cancers. Both agents generate cytotoxic double strand breaks (DSB) and abasic (apurinic/apyrimidinic (AP)) sites in DNA. The human AP endonuclease Ape1 acts on abasic or 3'-blocking DNA lesions such as those generated by IR or BLM. We examined the effect of siRNA-mediated Ape1 suppression on DNA repair and cellular resistance to IR or BLM in human B-lymphoblastoid TK6 cells and HCT116 colon tumor cells. Partial Ape1 deficiency (～30% of normal levels) sensitized cells more dramatically to BLM than to IR cytotoxicity. In both cases, expression of the unrelated yeast AP endonuclease, Apn1, largely restored resistance. Ape1 deficiency increased DNA AP site accumulation due to IR treatment but reduced the number of DSB. In contrast, for BLM, there were more DSB under Ape1 deficiency, with little change in the accumulation of AP sites. Although the role of Ape1 in generating DSB was greater for IR, the enzyme facilitated removal of AP sites, which may mitigate the cytotoxic effects of IR. In contrast, BLM generates scattered AP sites, and the DSB have 3'-phosphoglycolate termini that require Ape1 processing. These DSB persist under Ape1 deficiency. Apoptosis induced by BLM (but not by IR) under Ape1 deficiency was partially p53-dependent, more dramatically in TK6 than HCT116 cells. Thus, Ape1 suppression or inhibition may be a more efficacious adjuvant for BLM than for IR cancer therapy, particularly for tumors with a functional p53 pathway.     

Molecular and biological roles of Ape1 protein in mammalian base excision repair

Many oxidative DNA lesions are handled well by base excision repair (BER), but some types may be problematic. Recent work indicates that 2-deoxyribonolactone (dL) is such a lesion by forming stable, covalent cross-links between the abasic residue and DNA repair proteins with lyase activity. In the case of DNA polymerase beta, the reaction is potentiated by incision of dL by Ape1, the major mammalian AP endonuclease. When repair is prevented, polymerase beta is the most reactive cross-linking protein in whole-cell extracts. Cross-linking with dL is largely avoided by processing the damage through the "long-patch" (multinucleotide) BER pathway. However, if excess damage leads to the accumulation of unrepaired oxidative lesions in DNA, there may be a danger of polymerase beta-mediated cross-link formation. Understanding how cells respond to such complex damage is an important issue. In addition to its role in defending against DNA damage caused by exogenous agents, Ape1 protein is essential for coping with the endogenous DNA damage in human cells grown in culture. Suppression of Ape1 using RNA-interference technology causes arrest of cell proliferation and activation of apoptosis in various cell types, correlated with the accumulation of unrepaired abasic DNA damage. Notably, all these effects are reversed by expression of the unrelated protein Apn1 of S. cerevisiae, which shares only the enzymatic repair function with Ape1 (AP endonuclease).     

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1

Background

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases.

Methods

Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4^cat and UNG from different structural superfamilies were used.

Results

We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1.

Conclusions

We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4^cat, AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions.

General significance

Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.     

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.     

Action of human apurinic endonuclease (Ape1) on C1'-oxidized deoxyribose damage in DNA

Oxidative damage to DNA includes diverse lesions in the sugar-phosphate backbone. The chemical "nuclease" bis(1,10-phenanthroline)copper complex [(OP)(2)Cu] is believed to generate a mixture of direct oxidative strand breaks and C1'-oxidized abasic sites (2-deoxyribonolactone; dL). We found that, under our conditions, the lesions produced by (OP)(2)Cu (50 microM) in synthetic duplex DNA were predominantly dL, accompanied by approximately 30% direct strand breaks with 3'-phosphates. For enzymatic studies, (OP)(2)Cu was used to introduce damage with limited sequence-selectivity, while photolysis of a site-specific 2'-deoxyuridine-1'-t-butyl ketone generated dL at a defined position. The results showed that Ape1, the major human abasic endonuclease, catalyzed 5'-incision of dL sites, but acted at least 10-fold less effectively to remove the 3'-phosphates at direct strand breaks. Kinetic analysis of Ape1 incision using the site-specific dL substrate revealed the same k(cat) for dL and regular (glycosylase-generated) abasic sites, but with K(m) approximately five-fold higher for dL substrate. The efficiency of Ape1 acting on dL, and the abundance of this enzyme in vivo, indicate that dL sites in vivo would be rapidly processed by the endonuclease. The recent observation that Ape1-cleaved dL sites can covalently trap DNA polymerase beta during the abasic excision process suggests that efficient incision of dL by Ape1 may potentiate further problems in DNA repair.     

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.     

Mutational analysis of endonuclease V from Thermotoga maritima

Endonuclease V nicks damaged DNA at the second phosphodiester bond 3' to inosine, uracil, mismatched bases, or abasic (AP) sites. Alanine scanning mutagenesis was performed in nine conserved positions of Thermotoga maritima endonuclease V to identify amino acid residues involved in recognition or endonucleolytic cleavage of these diverse substrates. Alanine substitution at D43, E89, and D110 either abolishes or substantially reduces inosine cleavage activity. These three mutants gain binding affinity for binding to double-stranded or single-stranded inosine substrates in the absence of a metal ion, suggesting that these residues may be involved in coordinating catalytic metal ion(s). Y80A, H116A, and, to a lesser extent, R88A demonstrate reduced affinities for double-stranded or single-stranded inosine substrates or nicked products. The lack of tight binding to a nicked inosine product accounts for the increased rate of turnover of inosine substrate since the product release is less rate-limiting. Y80A, R88A, and H116A fail to cleave AP site substrates. Their activities toward uracil substrates are in the following order: H116A > R88A > Y80A. These residues may play a role in substrate recognition. K139A maintains wild-type binding affinity for binding to double-stranded and single-stranded inosine substrate, but fails to cleave AP site and uracil substrate efficiently, suggesting that K139 may play a role in facilitating non-inosine substrate cleavage.     

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.     

Elements in abasic site recognition by the major human and Escherichia coli apurinic/apyrimidinic endonucleases.

Sites of base loss in DNA arise spontaneously, are induced by damaging agents or are generated by DNA glycosylases. Repair of these potentially mutagenic or lethal lesions is carried out by apurinic/apyrimidinic (AP) endonucleases. To test current models of AP site recognition, we examined the effects of site-specific DNA structural modifications and an F266A mutation on incision and protein-DNA complex formation by the major human AP endonuclease, Ape. Changing the ring component of the abasic site from a neutral tetrahydrofuran (F) to a positively charged pyrrolidine had only a 4-fold effect on the binding capacity of Ape. A non-polar 4-methylindole base analog opposite F had a <2-fold effect on the incision activity of Ape and the human protein was unable to incise or specifically bind 'bulged' DNA substrates. Mutant Ape F266A protein complexed with F-containing DNA with only a 6-fold reduced affinity relative to wild-type protein. Similar studies are described using Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The results, in combination with previous findings, indicate that the ring structure of an AP site, the base opposite an AP site, the conformation of AP-DNA prior to protein binding and the F266 residue of Ape are not critical elements in targeted recognition by AP endonucleases.