Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.

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.

Endonuclease IV and T4 ligase enhanced detection of mutations in low abundance.

We propose a mutant detection approach based on endonuclease IV and DNA ligase in combination with qPCR. The enzymes functioned cooperatively to facilitate PCR for low abundance DNA detection. We demonstrate that our approach can distinguish mutations as low as 0.01%, indicating the potential application of this strategy in early cancer diagnosis.

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.

A universal probe system for low-abundance point mutation detection based on endonuclease IV.

Single base mutations are closely related to cancer diagnosis and treatment. The fluorescent probe method is one of the important methods to detect single-base mutations. We constructed a universal probe detection system based on endonuclease IV and the DNA strand displacement reaction. The system uses two toehold strand displacement reactions to relay the mutation information to the universal strand. There is no need to design the probe one-by-one for each mutation point during multi-site detection. It has the advantages of simple operation, rapid detection, and low cost. We used this method to detect common clinical mutation sites (PTEN R130Q/EGFR L858R/PTEN rs1473918395), and the detection limit can reach 0.1%-1%. The detection system can provide a new rapid and economical method for clinical single-base mutation detection, and has broad application prospects in diagnosis and prognostic evaluation.

Endonuclease IV-Regulated DNAzyme Motor for Universal Single-nucleotide Variation Discrimination.

Single-nucleotide variation (SNV) detection plays significant roles in disease diagnosis and treatment. Generally, auxiliary probe, restricted design rules, complicated detection system, and repeated experimental parameter optimization are needed to obtain satisfactory tradeoff between sensitivity and selectivity for SNV discrimination, especially when different mutant sites need to be distinguished. To overcome these limitations, we developed a universal, straightforward, and relatively cheap SNV discrimination strategy, which simultaneously possessed high sensitivity and selectivity. The excellent performance of this strategy was ascribed to the SNV discrimination property of endonuclease IV (Endo IV) and the different hydrolysis behavior between free deoxyribozyme (DNAzyme) and the trapped DNAzyme to the substrates modified on gold nanoparticles (AuNPs). When Endo IV recognized the mutant-type target (MT), free DNAzyme was released from the probe, and the DNAzyme motor was activated with the help of cofactor Mn2+ to generate an amplified fluorescence signal. On the contrary, the wild-type target (WT) could not effectively trigger the DNAzyme motor. Moreover, for different SNV types, the corresponding probe could be designed by simply changing the sequence hybridized with the target and retaining the DNAzyme sequence. Thus, the fluorescence signal generation system does not need to change for different SNV targets. Five clinical-related SNVs were determined with the limit of detection (LOD) ranging from 0.01 to 0.05%, which exhibited competitive sensitivity over existing SNV detection methods. This strategy provided another insight into the properties of Endo IV and DNAzyme, expanded the applications of DNAzyme motor, and has great potential to be used for precision medicine.

Guiding-Strand-Controlled DNA Nucleases with Enhanced Specificity and Tunable Kinetics for DNA Mutation Detection.

Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.

[Characterization of Recombinant Endonuclease IV from Mycobacterium tuberculosis].

Mycobacterium tuberculosis cells contain two apurinic/apyrimidinic (AP) endonucleases, endonuclease IV (MtbEnd) and exonuclease III (MtbXthA), the former playing a dominant role in protecting mycobacterial DNA from oxidative stress. Mycobacterial endonuclease IV substantially differs from its homologs found in Escherichia coli and other proteobacteria in a number of conserved positions important for DNA binding and AP site recognition. The M. tuberculosis end gene was cloned, and recombinant MtbEnd purified and characterized. The protein efficiently hydrolyzed DNA at the natural AP site and its 1'-deoxy analog in the presence of divalent cations, of which Ca^(2+), Mn^(2+), and Co^(2+) supported the highest activity. Exonuclease activity was not detected in MtbEnt preparations. The pH optimum was estimated at 7.0-8.0; the ionic strength optimum, at ~50 mM NaCl. Enzymatic activity of MtbEnd was suppressed in the presence of methoxyamine, a chemotherapeutic agent that modifies AP sites. Based on the results, MtbEnd was assumed to provide a possible target for new anti-tuberculosis drugs.

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.

Branch migration-based polymerase chain reaction combined with endonuclease IV-assisted target recycling probe/blocker system for detection of low-abundance point mutations.

Sensitive detection of low-abundance point mutations in blood or tissue may provide a great opportunity for the minimally invasive diagnosis of cancer and other related diseases. We demonstrate a novel method for ultra-sensitive detection of point mutations at low abundance by combination of branch migration-based PCR with endonuclease IV-assisted target recycling probe/blocker system. The method is able to identify the point mutations at abundances down to 0.01-0.02%. We anticipate this method to be widely adopted in clinical diagnosis and molecular research.

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.

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.

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.

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.

Base excision repair initiated rolling circle amplification-based fluorescent assay for screening uracil-DNA glycosylase activity using Endo IV-assisted cleavage of AP probes.

Uracil-DNA glycosylase (UDG) is a crucial damage repair enzyme that initiates the cellular base excision repair pathway that maintains the integrity of the genome. Abnormal UDG activity may induce the malfunction of uracil excision repair that is directly related to a range of diseases including cancers, genotypic diseases, and human immunodeficiencies. In this work, a simple, robust and cost effective biosensing platform for the ultrasensitive detection of UDG activity is established based on the combination of base excision repair-initiated primer generation for rolling circular amplification (RCA) with Endo IV-assisted signal amplification. In the presence of target UDG, UDG can catalyze the removal of uracil on a hairpin probe (HP) leaving an apurinic/apyrimidinic (AP site) which can be cleaved by Endo IV to generate a primer for triggering the RCA reaction. Subsequently, numerous AP site-embedded signal probes, acting as fluorescence-quenched probes, combine with the RCA products to perform signal transduction and quadradic signal amplification through an Endo IV-catalyzed cleavage reaction, thus significantly enhancing the fluorescence signal, which can be used for UDG activity screening. Under optimum conditions, this biosensor exhibits improved sensitivity toward target UDG with a detection limit of as low as 9.3 × 10-5 U mL-1 and a wide detection range across 5 orders of magnitude. Additionally, our biosensor demonstrates high selectivity toward UDG for simple, rapid, and low-cost detection. Furthermore, by redesigning the modification of HP and using of suitable endonuclease enzymes, this RCA coupled with Endo IV-assisted signal amplification strategy might be applied for the detection of various other targets, such as thymine DNA glycosylase, 8-oxoguanine DNA glycosylase, DNA methyltransferase, and so on. Hence, the proposed strategy provides a useful and versatile biosensing platform for the ultrasensitive detection of UDG activity and related fundamental biomedicine research and clinical diagnosis.

Thermococcus Eurythermalis Endonuclease IV Can Cleave Various Apurinic/Apyrimidinic Site Analogues in ssDNA and dsDNA.

Endonuclease IV (EndoIV) is a DNA damage-specific endonuclease that mainly hydrolyzes the phosphodiester bond located at 5' of an apurinic/apyrimidinic (AP) site in DNA. EndoIV also possesses 3'-exonuclease activity for removing 3'-blocking groups and normal nucleotides. Here, we report that Thermococcus eurythermalis EndoIV (TeuendoIV) shows AP endonuclease and 3'-exonuclease activities. The effect of AP site structures, positions and clustered patterns on the activity was characterized. The AP endonuclease activity of TeuendoIV can incise DNA 5' to various AP site analogues, including the alkane chain Spacer and polyethylene glycol Spacer. However, the short Spacer C2 strongly inhibits the AP endonuclease activity. The kinetic parameters also support its preference to various AP site analogues. In addition, the efficient cleavage at AP sites requires ≥2 normal nucleotides existing at the 5'-terminus. The 3'-exonuclease activity of TeuendoIV can remove one or more consecutive AP sites at the 3'-terminus. Mutations on the residues for substrate recognition show that binding AP site-containing or complementary strand plays a key role for the hydrolysis of phosphodiester bonds. Our results provide a comprehensive biochemical characterization of the cleavage/removal of AP site analogues and some insight for repairing AP sites in hyperthermophile cells.

Endonuclease IV cleaves apurinic/apyrimidinic sites in single-stranded DNA and its application for biosensing.

Endonuclease IV (Endo IV), as a DNA repairing enzyme, plays a crucial role in repairing damaged DNA comprising abasic sites to maintain genomic integrity. The cleaving capability of Endo IV to apurinic/apyrimidinic sites (AP) in single-stranded DNA (ssDNA) was demonstrated. It was found that Endo IV has considerably high cleaving activity to AP sites in ssDNA compared with that in double-stranded DNA (dsDNA). The unique feature of Endo IV in cleaving AP sites in ssDNA was further applied to construct a novel dual signal amplified sensing system for highly sensitive enzyme and protein detection by a combination of exonuclease III (Exo III)-aided cyclic amplification reaction and a rolling circle replication (RCR) technique, which showed a good sensing performance with a detection limit of 0.008 U mL(-1) for Endo IV and 2.5 pM for streptavidin. In addition, the developed method had considerably high specificity for Endo IV and streptavidin over other potential interferences. The developed strategy indeed provides a novel platform for protein and enzyme assays and may find a broad spectrum of applications in bioanalysis, disease diagnosis, and drug development.

A unique dual recognition hairpin probe mediated fluorescence amplification method for sensitive detection of uracil-DNA glycosylase and endonuclease IV activities.

Uracil-DNA glycosylase (UDG) and endonuclease IV (Endo IV) play cooperative roles in uracil base-excision repair (UBER) and inactivity of either will interrupt the UBER to cause disease. Detection of UDG and Endo IV activities is crucial to evaluate the UBER process in fundamental research and diagnostic application. Here, a unique dual recognition hairpin probe mediated fluorescence amplification method was developed for sensitively and selectively detecting UDG and Endo IV activities. For detecting UDG activity, the uracil base in the probe was excised by the target enzyme to generate an apurinic/apyrimidinic (AP) site, achieving the UDG recognition. Then, the AP site was cleaved by a tool enzyme Endo IV, releasing a primer to trigger rolling circle amplification (RCA) reaction. Finally, the RCA reaction produced numerous repeated G-quadruplex sequences, which interacted with N-methyl-mesoporphyrin IX to generate an enhanced fluorescence signal. Alternatively, for detecting Endo IV activity, the uracil base in the probe was first converted into an AP site by a tool enzyme UDG. Next, the AP site was cleaved by the target enzyme, achieving the Endo IV recognition. The signal was then generated and amplified in the same way as those in the UDG activity assay. The detection limits were as low as 0.00017 U mL(-1) for UDG and 0.11 U mL(-1) for Endo IV, respectively. Moreover, UDG and Endo IV can be well distinguished from their analogs. This method is beneficial for properly evaluating the UBER process in function studies and disease prognoses.

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.

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.