DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands

DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase–polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase–polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.     

Human AlkB homologue 1 (ABH1) exhibits DNA lyase activity at abasic sites

Bacterial AlkB and three human AlkB homologues (ABH1, ABH2, and ABH3) are Fe²⁺/2-oxoglutarate-dependent oxygenases that directly repair alkylation-damaged DNA. Here, we show that ABH1 unexpectedly has a second activity, cleaving DNA at abasic (AP) sites such as those arising spontaneously from alkylation-dependent depurination reactions. The DNA cleavage activity of ABH1 does not require added Fe²⁺ or 2-oxoglutarate, is not inhibited by EDTA, and is unaffected by mutation of the putative metal-binding residues, indicating that this activity arises from an active site distinct from that used for demethylation. AP-specific DNA cleavage was shown to occur by a lyase mechanism, rather than by hydrolysis, with the enzyme remaining associated with the DNA product. ABH1 can cleave at closely spaced AP-sites on opposite DNA strands yielding double-strand breaks in vitro and this reaction may relate to the physiological role of this unexpected AP lyase activity.     

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

The chemical stability of abasic RNA compared to abasic DNA

We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2′-O-protecting group together with the analogous DNA and the 2′-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2′-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37°C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via β,δ-elimination and 2′,3′-cyclophosphate formation. We found that β,δ-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only β,δ-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role.     

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.     

Cleavage of abasic sites in DNA by intercalator-amines

Anthraquinone and naphthalene diimide intercalators with amine-containing side chains cleave plasmid DNA at abasic sites (apurinic or apyrimidinic (AP) sites). The intercalator-amine is substantially more effective than the amine itself; many intercalators with diamine side chains cleave most of the abasic sites at micromolar concentration (30 min at 37 degrees C). Intercalators with two amino moieties in the side chain are more efficient than those with one, arguing for a role for each of two amines in the cleavage mechanism. Side chains ending in tertiary amines are somewhat more effective than those ending in primary amines, indicating that imine formation is not required for cleavage at the abasic site. We also report a systematic study of abasic site cleavage by polyamines, including piperidine, spermine, spermidine and 12 other di-, tri- and tetra-amines. For polyamines as well as intercalator-amines, examples with three carbon atoms between neighboring nitrogens atoms cleave most efficiently. This may reflect a particularly favorable geometry for proton abstraction for these species. The effect of nitrogen-nitrogen spacing on the pKa values of the nitrogens may contribute as well. Overall, cleavage of plasmid DNA at adventitious abasic sites by intercalator-amines bearing two nitrogens in a single side chain occurs readily.     

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.     

The rabbit beta-globin gene contains a large large insert in the coding sequence

We have used the rabbit β-globin DNA plasmid PβG1 (Maniatis et al., 1976) labeled with ³²P as a filter hybridization probe for DNA fragments containing the β-globin gene in restriction endonuclease digests of rabbit liver DNA. The β-globin DNA fragments we detect appear to contain the gene, present in PβG1 DNA, which codes for adult rabbit β-globin. These fragments have been ordered into a physical map of cleavage sites within and neighboring the structural gene in the rabbit genome (Jeffreys and Flavell, 1977). A detailed analysis of β-globin DNA fragments produced by cleavage with restriction endonucleases which are known to cut the β-globin gene has now shown that the β-globin structural gene is not contiguous in rabbit liver DNA, but is interrupted by a 600 base pair DNA segment inserted somewhere within the coding sequence for amino acid residues 101–120 of the 146 residue β-globin chain. Otherwise, the map of cleavage sites within the gene is co-linear with that deduced from the sequence of rabbit β-globin messenger RNA. Preliminary analysis indicates that this insert is also present in the β-globin gene in rabbit brain, kidney, spleen, bone marrow and sperm, and in erythroid cells isolated from the marrow of an anemic rabbit. The insert appears, therefore, to be a general property of the rabbit β-globin gene, even in tissues in which this gene is active, which suggests that the insert is not involved in inactivating the gene in nonerythroid tissues.     

Product inhibition and magnesium modulate the dual reaction mode of hOgg1

8-Oxoguanine (8-oxoG) is a major mutagenic DNA base damage corrected by the base excision repair (BER) pathway, which is initiated by lesion specific DNA glycosylases. The human DNA glycosylase hOgg1 catalyses excision of 8-oxoG followed by strand incision 3' to the abasic site if cytosine is positioned in the complementary strand. Unlike most bifunctional glycosylases, hOgg1 uncouples base removal and strand cleavage. This paper addresses the significance of product inhibition and magnesium for the non-concerted action of hOgg1 activities. The enzymatic activities of hOgg1 were analysed on duplex DNA containing a single 8-oxoG or abasic site opposite cytosine. AP-lyase cleavage of abasic sites was inhibited in the presence of free 8-oxoG, indicating that the product of base excision inhibits the subsequent strand incision step. Assays with DNA containing 8-oxoG showed that free 8-oxoG also inhibited the glycosylase activity. This result suggests that the free 8-oxoG base may retain in the recognition site following N-glycosylic cleavage, implying that product inhibition contribute to uncoupling the activities of hOgg1. Magnesium reduced the efficiency of base excision and strand incision on DNA containing 8-oxoG under single turnover conditions; however, the reduction was more pronounced for the AP-lyase activity. Furthermore, Shiff-base formation between hOgg1 and 8-oxoG containing DNA was abrogated in the presence of magnesium. These results suggest that hOgg1 mainly operates as a monofunctional glycosylase under physiological concentrations of magnesium.     

Cationic perylenediimide as a specific fluorescent binder to mismatch containing DNA

Small ligand molecules, which can recognize thermodynamically unstable site within DNA, such as mismatch base pair, abasic site, and single-bulge, have attracted much attention because of their potential diagnostics and biological applications. In this paper, we describe the binding of cationic perylenediimide (cPDI) molecules to thymine-containing mismatch base pair in DNA and the formation of cPDI dimer at the mismatch site. The cPDI dimer exhibits a characteristic excimer emission at 650 nm. For T/T mismatch containing DNA, the switching behavior from the PDI dimer (650 nm) to the monomer (550 nm) emission in specific response to Hg²⁺ ion was observed.     

DNA hydrolysis and oxidative cleavage by metal-binding peptides tethered to rhodium intercalators.

With the goal of developing artificial nucleases for DNA hydrolysis, metal-coordinating peptides have been tethered to a DNA-intercalating rhodium complex to deliver metal ions to the sugar-phosphate backbone. The intercalator, [Rh(phi)(2)bpy']Cl(3) [phi = 9,10-phenanthrenequinone diimine; bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine], provides DNA binding affinity, and a metal-binding peptide contributes reactivity. This strategy for DNA hydrolysis is a general one, and zinc(II)-promoted cleavage has been demonstrated for two widely different tethered metallopeptides. An intercalator coupled with a de novo-designed alpha helix containing two histidine residues has been demonstrated to cleave both supercoiled plasmid and linear DNA substrates. Mutation of this peptide confirms that the two histidine residues are essential for Zn(2+) binding and cleavage. Zinc(II)-promoted cleavage of supercoiled plasmid has also been demonstrated with an intercalator-peptide conjugate containing acidic residues and modeled after the active site of the BamHI endonuclease. Other redox-active metals, such as copper, have been delivered to DNA with our intercalator-peptide conjugates to effect oxidative chemistry. Copper cleavage experiments and photocleavage experiments with [Rh(phi)(2)bpy'](3+) complement the hydrolysis studies and provide structural information about the interactions between the tethered metallopeptides and DNA. Variation of the rhodium intercalator was also explored, but with a mismatch-specific intercalator, no site-specific hydrolysis was found. These experiments, in which the peptide, the metal cation, and the intercalator components of the conjugate are each varied, illustrate some of the issues involved in creating an artificial nuclease with DNA intercalators and metallopeptides.     

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N ²-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.     

Human AP-endonuclease (Ape1) activity on telomeric G4 structures is modulated by acetylatable lysine residues in the N-terminal sequence

Loss of telomeres stability is a hallmark of cancer cells. Exposed telomeres are prone to aberrant end-joining reactions leading to chromosomal fusions and translocations. Human telomeres contain repeated TTAGGG elements, in which the 3′ exposed strand may adopt a G-quadruplex (G4) structure. The guanine-rich regions of telomeres are hotspots for oxidation forming 8-oxoguanine, a lesion that is handled by the base excision repair (BER) pathway. One key player of this pathway is Ape1, the main human endonuclease processing abasic sites. Recent evidences showed an important role for Ape1 in telomeric physiology, but the molecular details regulating Ape1 enzymatic activities on G4-telomeric sequences are lacking. Through a combination of in vitro assays, we demonstrate that Ape1 can bind and process different G4 structures and that this interaction involves specific acetylatable lysine residues (i.e. K^27/31/32/35) present in the unstructured N-terminal sequence of the protein. The cleavage of an abasic site located in a G4 structure by Ape1 depends on the DNA conformation or the position of the lesion and on electrostatic interactions between the protein and the nucleic acids. Moreover, Ape1 mutants mimicking the acetylated protein display increased cleavage activity for abasic sites. We found that nucleophosmin (NPM1), which binds the N-terminal sequence of Ape1, plays a role in modulating telomere length and Ape1 activity at abasic G4 structures. Thus, the Ape1 N-terminal sequence is an important relay site for regulating the enzyme’s activity on G4-telomeric sequences, and specific acetylatable lysine residues constitute key regulatory sites of Ape1 enzymatic activity dynamics at telomeres.     

Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The DNA of all organisms is persistently damaged by endogenous reactive molecules. Most of the single-base endogenous damage is repaired through the base excision repair (BER) pathway that is initiated by members of the DNA glycosylase family. Although the BER pathway is often considered to proceed through a common abasic site intermediate, emerging evidence indicates that there are likely distinct branches reflected by the multitude of chemically different 3′ and 5′ ends generated at the repair site. In this study, we have applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS) to the analysis of model DNA substrates acted on by recombinant glycosylases. We examine the chemical identity of several possible abasic site and nicked intermediates generated by monofunctional and bifunctional glycosylases. Our results suggest that the intermediate from endoIII/Nth might not be a simple β-elimination product as described previously. On the basis of ¹⁸O incorporation experiments, we propose a new mechanism for the endoIII/Nth family of glycosylases that may resolve several of the previous controversies. We further demonstrate that the use of an array of lesion-containing oligonucleotides can be used to rapidly examine the substrate preferences of a given glycosylase. Some of the lesions examined here can be acted on by more than one glycosylase, resulting in a spectrum of damaged intermediates for each lesion, suggesting that the sequence and coordination of repair activities that act on these lesions may influence the biological outcome of damage repair.     

Long patch base excision repair compensates for DNA polymerase β inactivation by the C4'-oxidized abasic site

The C4'-oxidized abasic site (C4-AP), which is produced by a variety of damaging agents, has significant consequences for DNA. The lesion is highly mutagenic and reactive, resulting in interstrand cross-links. The base excision repair of DNA containing independently generated C4-AP was examined. C4-AP is incised by Ape1 ~12-fold less efficiently than an apurinic/apyrimidinic lesion. DNA polymerase β induces the β-elimination of incised C4-AP in ternary complexes, duplexes, and single-stranded substrate. However, excision from a ternary complex is most rapid. In addition, the lesion inactivates the enzyme after approximately seven turnovers on average by reacting with one or more lysine residues in the lyase active site. Unlike 5'-(2-phosphoryl-1,4-dioxobutane), which very efficiently irreversibly inhibits DNA polymerase β, the lesion is readily removed by strand displacement synthesis conducted by the polymerase in conjunction with flap endonuclease 1. DNA repair inhibition by C4-AP may be a partial cause of the cytotoxicity of drugs that produce this lesion.     

The adduct formation between the thioguanine-polyamine ligands and DNA with the AP site under UVA irradiated and non-irradiated conditions

The AP sites are representative of DNA damage and known as an intermediate in the base excision repair (BER) pathway which is involved in the repair of damaged nucleobases by reactive oxygen species, UVA irradiation, and DNA alkylating agents. Therefore, it is expected that the inhibition or modulation of the AP site repair pathway may be a new type of anticancer drug. In this study, we investigated the effects of the thioguanine-polyamine ligands (^SG-ligands) on the affinity and the reactivity for the AP site under UVA irradiated and non-irradiated conditions. The ^SG-ligands have a photo-reactivity with the A-F-C sequence where F represents a tetrahydrofuran AP site analogue. Interestingly, the ^SG-ligands promoted the β-elimination of the AP site followed by the formation of a covalent bond with the β-eliminated fragment without UVA irradiation.     

Improved measurement of dibenzo[a,l]pyrene-induced abasic sites by the aldehyde-reactive probe assay

Dibenzo[a,l]pyrene (DB[a,l]P) induces abundant amounts of depurinating adducts that spontaneously dissociate to form abasic sites in DNA. However, several previous studies that used the aldehyde-reactive probe (ARP) assay, could not verify abasic site formation by DB[a,l]P. Therefore, we examined whether a modification of the ARP assay would allow greater quantification of abasic sites. A previous study indicated that the abasic site quantification is improved by letting ARP trap the nascent abasic sites in cells, before extracting DNA for the assay. To test whether the addition of ARP to the DB[a,l]P–DNA adduct-forming reaction would improve abasic site quantification, we treated calf thymus DNA (0.625 mg/mL) with DB[a,l]P (80 μM) and 3-methylcholanthrene-treated rat liver microsomes with or without ARP (3 mM). The inclusion of ARP in the adduct-forming reaction resulted in significantly greater detection of abasic sites (62 lesions/10⁶ bp versus 3.7 lesions/10⁶ bp). DB[a,l]P also induces DNA strand breaks. The strand breaks may occur at abasic sites and by other mechanisms, such as oxidative damage. ARP/O-methoxyamine-abasic site conjugates are refractory to strand breakage, however, ARP or O-methoxyamine (3–10 mM) could only partially protect DB[a,l]P-induced DNA degradation, presumably by protecting the abasic sites, but not the other strand breaks. These results suggest that if DNA strand breakages occur at the abasic sites or at bases flanking them, and the fragments are lost during DNA extraction, abasic site estimation could be compromised. To obtain an independent line of evidence for abasic site formation in DB[a,l]P-treated cells, mouse Mβ16 fibroblasts were treated with DB[a,l]P and O-methoxyamine. O-Methoxyamine is known to potentiate cytotoxicity of abasic site-inducing chemicals by forming abasic site conjugates, which partially inhibits their repair. O-Methoxyamine was found to increase DB[a,l]P cytotoxicity in these cells, supporting the idea that DB[a,l]P formed abasic sites. In summary, the inclusion of ARP in the DB[a,l]P–DNA adduct-forming reaction traps and protects the nascent abasic sites, allowing an improved quantification of abasic sites.     

Role of AP‐endonuclease (Ape1) active site residues in stabilization of the reactant enzyme‐DNA complex

Apurinic/apyrimidinic endonuclease 1 (Ape1) is an important metal‐dependent enzyme in the base excision repair mechanism, responsible for the backbone cleavage of abasic DNA through a phosphate hydrolysis reaction. Molecular dynamics simulations of Ape1 complexed to its substrate DNA performed for models containing 1 or 2 Mg²⁺‐ions as cofactor located at different positions show a complex with 1 metal ion bound on the leaving group site of the scissile phosphate to be the most likely reaction‐competent conformation. Active‐site residue His309 is found to be protonated based on pKa calculations and the higher conformational stability of the Ape1‐DNA substrate complex compared to scenarios with neutral His309. Simulations of the D210N mutant further support the prevalence of protonated His309 and strongly suggest Asp210 as the general base for proton acceptance by a nucleophilic water molecule.     

Structural changes of an abasic site in duplex DNA affect noncovalent binding of the spin label ç

The influence of structural changes of an abasic site in duplex DNA on noncovalent and site-directed spin labeling (NC-SDSL) of the spin label ç were examined with electron paramagnetic resonance (EPR) spectroscopy. The binding affinities of ç to sixteen different DNA duplexes containing all possible sequences immediately flanking the abasic site were determined and the results showed that the binding of ç is highly flanking-sequence dependent. In general, a 5′-dG nucleotide favors the binding of the spin label. In particular, 5′-d(G__T) was the best binding sequence whereas 5′-d(C__T) showed the lowest affinity. Changing the structure of the abasic site linker from a tetrahydrofuran analog (F) to the anucleosidic C₃-spacer (C₃) does not appreciably affect the binding of ç to the abasic site. For efficient binding of ç, the abasic site needs to be located at least four base pairs away from the duplex end. Introducing a methyl substituent at N3 of ç did not change the binding affinity, but a decreased binding was observed for both N3-ethyl and -propyl groups. These results will guide the design of abasic site receptors and spin label ligands for NC-SDSL of nucleic acids.