Purification, crystallization and preliminary crystallographic analysis of Chlamydophila pneumoniae AP endonuclease IV.

Base excision is a crucial DNA repair process mediated by endonuclease IV in nucleotide excision. In Chlamydia pneumoniae, CpendoIV is the exclusive AP endonuclease IV, exhibiting DNA replication error-proofreading capabilities, making it a promising target for anti-chlamydial drug development. Predicting the structure of CpendoIV, molecular docking with DNA was performed, analyzing complex binding sites and protein surface electrostatic potential. Comparative structural studies were conducted with E. coli EndoIV and DNA complex containing AP sites.CpendoIV was cloned, expressed in E. coli, and purified via Ni-NTA chelation and size-exclusion chromatography. Low NaCl concentrations induced aggregation during purification, while high concentrations enhanced purity.CpendoIV recognizes and cleaving AP sites on dsDNA, and Zn2+ influences the activity. Crystallization was achieved under 8% (v/v) Tacsimate pH 5.2, 25% (w/v) polyethylene glycol 3350, and 1.91 Å resolution X-ray diffraction data was obtained at 100 K. This research is significant for provides a deeper understanding of CpendoIV involvement in the base excision repair process, offering insights into Chlamydia pneumoniae.

Cloning and characterization of the major AP endonuclease from Staphylococcus aureus.

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Complete genome analysis of Staphylococcus aureus identified a single AP endonuclease, SaNfo, which is a member of the endonuclease IV family exemplified by Escherichia coli Nfo. At present, it remains unknown whether SaNfo possesses DNA repair activities similar to its counterparts from E. coli and other bacteria. Here, we report that the purified SaNfo protein contains efficient AP endonuclease and nucleotide incision repair (NIR) activities. Optimal reaction conditions for SaNfo-catalysed AP endonuclease activity are high ionic strength and Mn2+ concentration, pH in range 7.5-9.0 and the temperature optimum of 37-45 °C. Cell-free extracts of S. aureus exhibited efficient AP site cleavage and NIR activities. Heterologous expression of SaNfo strongly reduces the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2. Site-directed mutagenesis showed that the Glu258 residue is critical for the SaNfo enzyme function. The AP endonuclease but not the NIR activity of SaNfo were stimulated by the ß-clamp (SaDnaN dimer), suggesting that it might participate in the organization of BER in S. aureus. Overall, our data confirm that the activity, substrate specificity and in vivo functionality of S. aureus Nfo are consistent with this protein being the major AP endonuclease for the repair of DNA damage generated by endogenous and host-imposed factors.

In vitro eradication of abasic site-mediated DNA-peptide/protein cross-links by Escherichia coli long-patch base excision repair.

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA-protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5'-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5'-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2-36.1 kDa) DPCs is less efficient than that of an AP-peptide10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.

Integration of single-molecule detection with endonuclease IV-assisted signal amplification for sensitive DNA methylation assay.

We demonstrate the development of a new fluorescent biosensor for sensitive DNA methylation assay by integrating single-molecule detection with endo IV-assisted signal amplification. This biosensor possesses the characteristics of good selectivity and high sensitivity with a detection limit of 7.3 × 10-17 M. It can distinguish as low as 0.01% methylation level, and can analyze genomic DNA methylation even in a single cancer cell.

Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA walker and hybridization chain reaction amplification.

An ultrasensitive fluorescence sensing strategy for kanamycin (KANA) determination using endonuclease IV (Endo IV)-powered DNA walker, and hybridization chain reaction (HCR) amplification was reported. The sensing system consists of Endo IV-powered 3D DNA walker using for the specific recognition of KANA and the formation of the initiators, two metastable hairpin probes as the substrates of HCR and a tetrahydrofuran abasic site (AP site)-embeded fluorescence-quenched probe for fluorescence signal output. On account of this skilled design of sensing system, the specific binding between KANA and its aptamer activates DNA walker, in which the swing arm can move autonomously along the 3D track via Endo IV-mediated hydrolysis of the anchorages, inducing the formation of initiators that initiates HCR and the following Endo IV-assisted cyclic cleavage of fluorescence reporter probes. The use of Endo IV offers the advantages of simplified and accessible design without the need of specific sequence in DNA substrates. Under the optimal experimental conditions, the fluorescence biosensor shows excellent sensitivity toward KANA detection with a detection limit as low as 1.01 pM (the excitation wavelength is 486 nm). The practical applicability of this strategy is demonstrated by detecting KANA in spiked milk samples with recovery in the range of 98 to 102%. Therefore, this reported strategy might create an accurate and robust fluorescence sensing platform for trace amounts of antibiotic residues determination and related safety analysis. Graphical abstract Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA Walker and hybridization chain, reaction amplification, Xiaonan Qu, Jingfeng Wang, Rufeng Zhang, Yihan Zhao, Shasha Li, Yu Wang, Su Liu*, Jiadong Huang, and Jinghua Yu, an ultrasensitive fluorescence sensing strategy for kanamycin determination using endonuclease IV-powered DNA walker, and hybridization chain reaction amplification is reported.

Proximity-enabled bidirectional enzymatic repairing amplification for ultrasensitive fluorescence sensing of adenosine triphosphate.

A novel fluorescence sensing strategy for ultrasensitive and highly specific detection of adenosine triphosphate (ATP) has been developed by the combination of the proximity ligation assay with bidirectional enzymatic repairing amplification (BERA). The strategy relies on proximity binding-triggered the release of palindromic tail that initiates bidirectional cyclic enzymatic repairing amplification reaction with the aid of polymerase and two DNA repairing enzymes, uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV). A fluorescence-quenched hairpin probe with a palindromic tail at the 3' end is skillfully designed that functions as not only the recognition element, primer, and polymerization template for BERA but also the indicator for fluorescence signal output. On the basis of the amplification strategy, this biosensor displays excellent sensitivity and selectivity for ATP detection with an outstanding detection limit of 0.81 pM. Through simultaneously enhancing the target response signal value and reducing nonspecific background, this work deducted the background effect, and showed high sensitivity and reproducibility. Moreover, our biosensor also shows promising potential in real sample analysis. Therefore, the proximity-enabled BERA strategy indeed creates a simple and valuable fluorescence sensing platform for ATP identification and related disease diagnosis and biomedical research.

Depurination of Colibactin-Derived Interstrand Cross-Links.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

Eliminating the secondary structure of targeting strands for enhancement of DNA probe based low-abundance point mutation detection.

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.

Primer remodeling amplification-activated multisite-catalytic hairpin assembly enabling the concurrent formation of Y-shaped DNA nanotorches for the fluorescence assay of ochratoxin A.

DNA can be configured into unique high-order structures due to its significantly high programmability, such as a three-way junction-based structure (denoted Y-shaped DNA), for further applications. Herein, we report a label-free fluorescent signal-on biosensor based on the target-driven primer remodeling rolling circle amplification (RCA)-activated multisite-catalytic hairpin assembly (CHA) enabling the concurrent formation of Y-shaped DNA nanotorches (Y-DNTs) for ultrasensitive detection of ochratoxin A (OTA). Two kinds of masterfully-designed probes, termed Complex I and II, were pre-prepared by the combination of a circular template (CT) with an OTA aptamer (S1), a substrate probe (S2) and hairpin probe 1 (HP1), respectively. Target OTA specifically binds to Complex I, resulting in the release of the remnant element in S2 and successive remodeling into a mature primer for RCA by phi29 DNA polymerase, thus a usable primer-CT complex is produced, which actuates primary RCA. Then, numerous Complex II probes can anneal with the first-generation RCA product (RP) with multiple sites to activate the CHA process. With the participation of endonuclease IV (Endo IV) and phi29, HP1 as a pre-primer containing a tetrahydrofuran abasic site mimic (AP site) in Complex II is converted into a mature primer to initiate additional rounds of RCA. So, countless Y-DNTs are formed concurrently containing a G-quadruplex structure that enables the N-methylmesoporphyrin IX (NMM) to be embedded, generating remarkably strong fluorescence signals. The biosensor was demonstrated to enable rapid and accurate highly efficient and selective detection of OTA with an improved detection limit of as low as 0.0002 ng mL-1 and a widened dynamic range of over 4 orders of magnitude. Meanwhile, this method was proven to be capable of being used to analyze actual samples. Therefore, this proposed strategy may be established as a useful and practical platform for the ultrasensitive detection of mycotoxins in food safety testing.

Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase.

DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies. Thus, the detection of DG activity is essential for biomedical research and clinical diagnosis. Herein, we develop a facile fluorometric method based on the base excision repair (BER) mediated cascading triple-signal amplification for the sensitive detection of DG. The presence of human alkyladenine DNA glycosylase (hAAG) can initiate the cleavage of the substrate at the mismatched deoxyinosine site by endonuclease IV (Endo IV), resulting in the breaking of the DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate strand displacement amplification (SDA) to release primers. The released primers can further bind to a circular template to induce an exponential primer generation rolling circle amplification (PG-RCA) reaction, producing a large number of primers. The primers that resulted from the SDA and PG-RCA reaction can induce the subsequent recycling cleavage of signal probes, leading to the generation of a fluorescence signal. Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification, this method exhibits a low detection limit of 0.026 U mL-1 and a large dynamic range of 4 orders of magnitude for hAAG. Moreover, this method has distinct advantages of simplicity and low cost, and it can further quantify the hAAG activity from HeLa cell extracts, holding great potential in clinical diagnosis and biomedical research.

Detection of Inosine on Transfer RNAs without a Reverse Transcription Reaction.

Inosine at the "wobble" position (I34) is one of the few essential posttranscriptional modifications in tRNAs (tRNAs). It results from the deamination of adenosine and occurs in bacteria on tRNAArgACG and in eukarya on six or seven additional tRNA substrates. Because inosine is structurally a guanosine analogue, reverse transcriptases recognize it as a guanosine. Most methods used to examine the presence of inosine rely on this phenomenon and detect the modified base as a change in the DNA sequence that results from the reverse transcription reaction. These methods, however, cannot always be applied to tRNAs because reverse transcription can be compromised by the presence of other posttranscriptional modifications. Here we present SL-ID (splinted ligation-based inosine detection), a reverse transcription-free method for detecting inosine based on an I34-dependent specific cleavage of tRNAs by endonuclease V, followed by a splinted ligation and polyacrylamide gel electrophoresis analysis. We show that the method can detect I34 on different tRNA substrates and can be applied to total RNA derived from different species, cell types, and tissues. Here we apply the method to solve previous controversies regarding the modification status of mammalian tRNAArgACG.

Integration of single-molecule detection with magnetic separation for multiplexed detection of DNA glycosylases.

We combine single-molecule detection with magnetic separation for simultaneous measurement of human 8-oxoG DNA glycosylase 1 (hOGG1) and uracil DNA glycosylase (UDG) based on excision repair-initiated endonuclease IV (Endo IV)-assisted signal amplification. This method can sensitively detect multiple DNA glycosylases, and it can be further applied for the simultaneous measurement of enzyme kinetic parameters and screening of both hOGG1 and UDG inhibitors.

Structure of D-tagatose 3-epimerase-like protein from Methanocaldococcus jannaschii.

The crystal structure of a D-tagatose 3-epimerase-like protein (MJ1311p) encoded by a hypothetical open reading frame, MJ1311, in the genome of the hyperthermophilic archaeon Methanocaldococcus jannaschii was determined at a resolution of 2.64 Å. The asymmetric unit contained two homologous subunits, and the dimer was generated by twofold symmetry. The overall fold of the subunit proved to be similar to those of the D-tagatose 3-epimerase from Pseudomonas cichorii and the D-psicose 3-epimerases from Agrobacterium tumefaciens and Clostridium cellulolyticum. However, the situation at the subunit-subunit interface differed substantially from that in D-tagatose 3-epimerase family enzymes. In MJ1311p, Glu125, Leu126 and Trp127 from one subunit were found to be located over the metal-ion-binding site of the other subunit and appeared to contribute to the active site, narrowing the substrate-binding cleft. Moreover, the nine residues comprising a trinuclear zinc centre in endonuclease IV were found to be strictly conserved in MJ1311p, although a distinct groove involved in DNA binding was not present. These findings indicate that the active-site architecture of MJ1311p is quite unique and is substantially different from those of D-tagatose 3-epimerase family enzymes and endonuclease IV.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once.

To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency.

Endonuclease IV Is the major apurinic/apyrimidinic endonuclease in Mycobacterium tuberculosis and is important for protection against oxidative damage.

During the establishment of an infection, bacterial pathogens encounter oxidative stress resulting in the production of DNA lesions. Majority of these lesions are repaired by base excision repair (BER) pathway. Amongst these, abasic sites are the most frequent lesions in DNA. Class II apurinic/apyrimidinic (AP) endonucleases play a major role in BER of damaged DNA comprising of abasic sites. Mycobacterium tuberculosis, a deadly pathogen, resides in the human macrophages and is continually subjected to oxidative assaults. We have characterized for the first time two AP endonucleases namely Endonuclease IV (End) and Exonuclease III (XthA) that perform distinct functions in M.tuberculosis. We demonstrate that M.tuberculosis End is a typical AP endonuclease while XthA is predominantly a 3'→5' exonuclease. The AP endonuclease activity of End and XthA was stimulated by Mg(2+) and Ca(2+) and displayed a preferential recognition for abasic site paired opposite to a cytosine residue in DNA. Moreover, End exhibited metal ion independent 3'→5' exonuclease activity while in the case of XthA this activity was metal ion dependent. We demonstrate that End is not only a more efficient AP endonuclease than XthA but it also represents the major AP endonuclease activity in M.tuberculosis and plays a crucial role in defense against oxidative stress.

Insight into mechanisms of 3'-5' exonuclease activity and removal of bulky 8,5'-cyclopurine adducts by apurinic/apyrimidinic endonucleases.

8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases--Escherichia coli Xth and human APE1--can remove 5'S cdA (S-cdA) at 3' termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3' end. To understand the structural basis of 3' repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2'-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3'-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3'→5' exonuclease function and its inhibition by 3'-terminal S-cdA residue. This structure was used as a template to model 3'-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3' repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3' DNA termini in E. coli and human cells, respectively.

Combination of a modified block PCR and endonuclease IV-based signal amplification system for ultra-sensitive detection of low-abundance point mutations.

By combination of a modified block PCR and endonuclease IV-based signal amplification system, we have developed a novel approach for ultra-sensitive detection of point mutations. The method can effectively identify mutant target sequence immersed in a large background of wild-type sequences with abundance down to 0.03% (for C→A) and 0.005% (for C→G). This sensitivity is among the highest in comparison with other existing approaches and the operating procedures are simple and time saving. The method holds great potential for future application in clinical diagnosis and biomedical research.

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 Mg(2+) 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 Mg(2+). 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.

The role of the PHP domain associated with DNA polymerase X from Thermus thermophilus HB8 in base excision repair.

Base excision repair (BER) is one of the most commonly used DNA repair pathways involved in genome stability. X-family DNA polymerases (PolXs) play critical roles in BER, especially in filling single-nucleotide gaps. In addition to a polymerase core domain, bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain with phosphoesterase activity which is also required for BER. However, the role of the PHP domain of PolX in bacterial BER remains unresolved. We found that the PHP domain of Thermus thermophilus HB8 PolX (ttPolX) functions as two types of phosphoesterase in BER, including a 3'-phosphatase and an apurinic/apyrimidinic (AP) endonuclease. Experiments using T. thermophilus HB8 cell lysates revealed that the majority of the 3'-phosphatase and AP endonuclease activities are attributable to the another phosphoesterase in T. thermophilus HB8, endonuclease IV (ttEndoIV). However, ttPolX possesses significant 3'-phosphatase activity in ΔttendoIV cell lysate, indicating possible complementation. Our experiments also reveal that there are only two enzymes that display the 3'-phosphatase activity in the T. thermophilus HB8 cell, ttPolX and ttEndoIV. Furthermore, phenotypic analysis of ΔttpolX, ΔttendoIV, and ΔttpolX/ΔttendoIV using hydrogen peroxide and sodium nitrite supports the hypothesis that ttPolX functions as a backup for ttEndoIV in BER.

DNA sequence context conceals α-anomeric lesions.

DNA sequence context has long been known to modulate detection and repair of DNA damage. Recent studies using experimental and computational approaches have sought to provide a basis for this observation. We have previously shown that an α-anomeric adenosine (αA) flanked by cytosines (5'CαAC-3') resulted in a kinked DNA duplex with an enlarged minor groove. Comparison of different flanking sequences revealed that a DNA duplex containing a 5'CαAG-3' motif exhibits unique substrate properties. However, this substrate was not distinguished by unusual thermodynamic properties. To understand the structural basis of the altered recognition, we have determined the solution structure of a DNA duplex with a 5'CαAG-3' core, using an extensive set of restraints including dipolar couplings and backbone torsion angles. The NMR structure exhibits an excellent agreement with the data (total R(X) <5.3%). The αA base is intrahelical, in a reverse Watson-Crick orientation, and forms a weak base pair with a thymine of the opposite strand. In comparison to the DNA duplex with a 5'CαAC-3' core, we observe a significant reduction of the local perturbation (backbone, stacking, tilt, roll, and twist), resulting in a straighter DNA with narrower minor groove. Overall, these features result in a less perturbed DNA helix and obscure the presence of the lesion compared to the 5'CαAC-3' sequence. The improved stacking of the 5'CαAG-3' core also affects the energetics of the DNA deformation that is required to form a catalytically competent complex. These traits provide a rationale for the modulation of the recognition by endonuclease IV.