Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroimindiohydantoin and guanidinohydantoin

Base excision repair is the major pathway for removal of oxidative DNA base damage. This pathway is initiated by DNA glycosylases, which recognize and excise damaged bases from DNA. In this work, we have purified the glycosylase domain (GD) of human DNA glycosylase NEIL3. The substrate specificity has been characterized and we have elucidated the catalytic mechanisms. GD NEIL3 excised the hydantoin lesions spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) in single-stranded (ss) and double-stranded (ds) DNA efficiently. NEIL3 also removed 5-hydroxy-2′-deoxycytidine (5OHC) and 5-hydroxy-2′-deoxyuridine (5OHU) in ssDNA, but less efficiently than hydantoins. Unlike NEIL1 and NEIL2, which possess a β,δ-elimination activity, NEIL3 mainly incised damaged DNA by β-elimination. Further, the base excision and strand incision activities of NEIL3 exhibited a non-concerted action, indicating that NEIL3 mainly operate as a monofunctional DNA glycosylase. The site-specific NEIL3 mutant V2P, however, showed a concerted action, suggesting that the N-terminal amino group in Val2 is critical for the monofunctional modus. Finally, we demonstrated that residue Lys81 is essential for catalysis.     

Genome and cancer single nucleotide polymorphisms of the human NEIL1 DNA glycosylase: Activity,structure, and the effect of editing

The repair of free-radical oxidative DNA damage is carried out by lesion-specific DNA glycosylases as the first step of the highly conserved base excision repair (BER) pathway. In humans, three orthologs of the prototypical endonuclease VIII (Nei), the Nei-like NEIL1-3 enzymes are involved in the repair of oxidized DNA lesions. In recent years, several genome and cancer single-nucleotide polymorphic variants of the NEIL1 glycosylase have been identified. In this study we characterized four variants of human NEIL1: S82C, G83D, P208S, and ΔE28, and tested their ability to excise pyrimidine-derived lesions such as thymine glycol (Tg), 5-hydroxyuracil (5-OHU), and dihydrouracil (DHU) and the purine-derived guanidinohydantoin (Gh), spiroiminodihydantoin 1 (Sp1), and methylated 2,6-diamino-4-hydroxy-5-formamidopyrimidine (MeFapyG). The P208S variant has near wild-type activity on all substrates tested. The S82C and ΔE28 variants exhibit decreased Tg excision compared to wild-type. G83D displays little to no activity with any of the substrates tested, with the exception of Gh and Sp1. Human NEIL1 is known to undergo editing whereby the lysine at position 242 is recoded into an arginine. The non-edited form of NEIL1 is more efficient at cleaving Tg than the R242 form, but the G83D variant does not cleave Tg regardless of the edited status of NEIL1. The corresponding G86D variant in Mimivirus Nei1 similarly lacks glycosylase activity. A structure of a G86D–DNA complex reveals a rearrangement in the β4/5 loop comprising Leu84, the highly-conserved void-filling residue, thereby providing a structural rationale for the decreased glycosylase activity of the glycine to aspartate variant.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

Structural investigation of a viral ortholog of human NEIL2/3 DNA glycosylases

Assault to DNA that leads to oxidative base damage is repaired by the base excision repair (BER) pathway with specialized enzymes called DNA glycosylases catalyzing the first step of this pathway. These glycosylases can be categorized into two families: the HhH superfamily, which includes endonuclease III (or Nth), and the Fpg/Nei family, which comprises formamidopyrimidine DNA glycosylase (or Fpg) and endonuclease VIII (or Nei). In humans there are three Nei-like (NEIL) glycosylases: NEIL1, 2, and 3. Here we present the first crystal structure of a viral ortholog of the human NEIL2/NEIL3 proteins, Mimivirus Nei2 (MvNei2), determined at 2.04 Å resolution. The C-terminal region of the MvNei2 enzyme comprises two conserved DNA binding motifs: the helix-two-turns-helix (H2TH) motif and a C-H-C-C type zinc-finger similar to that of human NEIL2. The N-terminal region of MvNei2 is most closely related to NEIL3. Like NEIL3, MvNei2 bears a valine at position 2 instead of the usual proline and it lacks two of the three conserved void-filling residues present in other members of the Fpg/Nei family. Mutational analysis of the only conserved void-filling residue methionine 72 to alanine yields an MvNei2 variant with impaired glycosylase activity. Mutation of the adjacent His73 causes the enzyme to be more productive thereby suggesting a plausible role for this residue in the DNA lesion search process.     

Molecular characterization of a putative plant homolog of MBD4 DNA glycosylase

Methyl-CpG-binding domain 4 (MBD4) DNA glycosylase is involved in excision of spontaneous deamination products of cytosine and 5-methylcytosine in animals, but it is unknown whether related proteins perform similar functions in plants. We report here the isolation and biochemical characterization of a putative MBD4 homolog from Arabidopsis thaliana, designated as MBD4L (MBD4-like). The plant enzyme lacks the MBD domain present in mammalian MBD4 proteins, but conserves a DNA glycosylase domain with critical residues for substrate recognition and catalysis, and it is more closely related to MBD4 homologs than to other members of the HhH-GPD superfamily. Arabidopsis MBD4L excises uracil and thymine opposite G, and the presence of halogen substituents at C5 of the target base greatly increases its excision efficiency. No significant activity is detected on cytosine derivatives such as 5-methylcytosine or 5-hydroxymethylcytosine. The enzyme binds to the abasic site product generated after excision, which decreases its catalytic turnover in vitro. Both the full-length protein and a N-terminal truncated version retaining the catalytic domain exhibit a preference for a CpG sequence context, where most plant DNA methylation is found. Our results suggest that an important function of Arabidopsis MBD4L is to protect the plant genome from the mutagenic consequences of cytosine and 5-methylcytosine deamination.     

火球菌8-氧鸟嘌呤DNA糖苷酶基因克隆、表达纯化和酶学性质研究

【目的】在大肠杆菌中表达火球菌8-氧鸟嘌呤DNA糖苷酶,纯化得到重组火球菌8-氧鸟嘌呤DNA糖苷酶,在此基础上系统研究火球菌8-氧鸟嘌呤DNA糖苷酶的酶学特征。【方法】构建8-氧鸟嘌呤DNA糖苷酶重组表达质粒,将重组质粒转化Escherichia coli Rosetta(DE3),利用IPTG诱导表达重组蛋白,通过Ni2+亲和层析柱纯化重组蛋白;最后利用含8-氧鸟嘌呤损伤的寡核苷酸作为底物,测定8-氧鸟嘌呤DNA糖苷酶的酶学性质。【结果】在大肠杆菌中成功诱导表达了重组火球菌8-氧鸟嘌呤DNA糖苷酶,经Ni2+亲和纯化后蛋白纯度大于95%。在体外鉴定了重组火球菌8-氧鸟嘌呤DNA糖苷酶的酶学性质。结果表明重组火球菌8-氧鸟嘌呤DNA糖苷酶可以切除DNA中的8-氧鸟嘌呤(8-Oxo-G,GO)损伤碱基,并且具有AP裂解酶活性。重组火球菌8-氧鸟嘌呤DNA糖苷酶催化反应的最适pH值和温度分别是pH 8.5和55°C。除Zn2+对火球菌8-氧鸟嘌呤DNA糖苷酶的酶促反应有明显的抑制作用外,实验中测定的其它二价离子(Mn2+,Mg2+,Ca2+,Ni2+,Co2+,Cu2+)对其没有明显的影响。离子强度在50-100 mmol/L范围内对其酶促反应影响不大,超过100 mmol/L时有明显的抑制作用。与8-氧鸟嘌呤互补的碱基差异对火球菌8-氧鸟嘌呤DNA糖苷酶切除8-氧鸟嘌呤损伤的效率影响不大;但与单链DNA相比,双链DNA是优选底物,切割效率如下:GO/C≈GO/G≈GO/T≈GO/AGO/-。【结论】在大肠杆菌中成功表达,并Ni2+亲和纯化了火球菌8-氧鸟嘌呤DNA糖苷酶,生化研究表明制备的重组蛋白具有8-氧鸟嘌呤DNA糖苷酶活性,可能负责切除火球菌基因组DNA中的8-氧鸟嘌呤损伤。     

Processing of N5-substituted formamidopyrimidine DNA adducts by DNA glycosylases NEIL1 and NEIL3

A variety of agents cause DNA base alkylation damage, including the known hepatocarcinogen aflatoxin B₁ (AFB₁) and chemotherapeutic drugs derived from nitrogen mustard (NM). The N7 site of guanine is the primary site of alkylation, with some N7-deoxyguanosine adducts undergoing imidazole ring-opening to stable mutagenic N⁵-alkyl formamidopyrimidine (Fapy-dG) adducts. These adducts exist as a mixture of canonical β- and unnatural α-anomeric forms. The β species are predominant in double-stranded (ds) DNA. Recently, we have demonstrated that the DNA glycosylase NEIL1 can initiate repair of AFB₁-Fapy-dG adducts both in vitro and in vivo, with Neil1^−/− mice showing an increased susceptibility to AFB₁-induced hepatocellular carcinoma.Here, we hypothesized that NEIL1 could excise NM-Fapy-dG and that NEIL3, a closely related DNA glycosylase, could excise both NM-Fapy-dG and AFB₁-Fapy-dG. Product formation from the reaction of human NEIL1 with ds oligodeoxynucleotides containing a unique NM-Fapy-dG followed a bi-component exponential function under single turnover conditions. Thus, two adduct conformations were differentially recognized by hNEIL1. The excision rate of the major form (∼13.0 min⁻¹), presumed to be the β-anomer, was significantly higher than that previously reported for 5-hydroxycytosine, 5-hydroxyuracil, thymine glycol (Tg), and AFB₁-Fapy-dG. Product generation from the minor form was much slower (∼0.4 min⁻¹), likely reflecting the rate of conversion of the α anomer into the β anomer. Mus musculus NEIL3 (MmuNEIL3Δ324) excised NM-Fapy-dG from single-stranded (ss) DNA (turnover rate of ∼0.4 min⁻¹), but not from ds DNA. Product formation from ss substrate was incomplete, presumably because of a substantial presence of the α anomer. MmuNEIL3Δ324 could not initiate repair of AFB₁-Fapy-dG in either ds or ss DNA. Overall, the data suggest that both NEIL1 and NEIL3 may protect cells against cytotoxic and mutagenic effects of NM-Fapy-dG, but NEIL1 may have a unique role in initiation of base excision repair of AFB₁-Fapy-dG.     

Unique Structural Features of Mammalian NEIL2 DNA Glycosylase Prime Its Activity for Diverse DNA Substrates and Environments

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An aza-nucleoside,fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG)

The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG.     

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G^o) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar–phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5′ to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9–Rad1–Hus1 (9–1–1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein–DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.     

极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展

高温会加快碱基脱氨基反应形成损伤碱基的速率,进一步对脱氨基的碱基进行复制会导致突变。因此,极端嗜热古菌基因组的稳定性面临着其生存高温环境的挑战。胞嘧啶脱氨基形成尿嘧啶,是常见的脱碱基类型,复制DNA中尿嘧啶会造成GC→AT的突变。尿嘧啶DNA糖苷酶(Uracil DNA glycosylase,UDG)是修复DNA中尿嘧啶的关键酶。基于识别底物的特异性,UDG分为6个家族,广泛分布在细菌、古菌、真核生物以及一些病毒中。基因组序列显示,极端嗜热古菌至少编码一种UDG。目前,对于细菌和真核生物的UDG已进行了大量的研究,但是关于极端嗜热古菌UDG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌UDG的研究进展,并对今后的研究提出了展望。     

Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.     

Selective base excision repair of DNA damage by the non‐base‐flipping DNA glycosylase AlkC

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT‐like repeat (HLR) fold. AlkD uses a unique non‐base‐flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3‐methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non‐base‐flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin‐like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3‐methylcytosine (3mC) and N1‐methyladenine (1mA), which are also repaired by AlkB‐catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.     

Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli (E. coli) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant (K_D) of 0.25 nM. Steady-state enzyme kinetics showed an apparent K_m of 5.3 nM and k_cat of 0.2 min⁻¹ of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100 mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50 °C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure–function analysis.     

NEIL3-Dependent Regulation of Cardiac Fibroblast Proliferation Prevents Myocardial Rupture

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Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo

The 3,N(4)-ethenocytosine (epsilon C) residue might have biological role in vivo since it is recognized and efficiently excised in vitro by the E. coli mismatch-specific uracil-DNA glycosylase (MUG) and the human thymine-DNA glycosylase (hTDG). In the present work we have generated mug defective mutant of E. coli by insertion of a kanamycin cassette to assess the role of MUG in vivo. We show that human TDG complements the enzymatic activity of MUG when expressed in a mug mutant. The epsilon C-DNA glycosylase defective strain did not exhibit spontaneous mutator phenotype and did not show unusual sensitivity to any of the following DNA damaging treatments: methylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light, H(2)O(2), paraquat. However, plasmid DNA damaged by 2-chloroacetaldehyde treatment in vitro was inactivated at a greater rate in a mug mutant than in wild-type host, suggesting that MUG is required for the in vivo processing of the ethenobases. In addition, 2-chloroacetaldehyde treatment induces preferentially G.C --> C.G and A.T --> T.A transversions in mug mutant. Comparison of the mutation frequencies induced by the site-specifically incorporated epsilon C residue in E. coli wild-type versus mug indicates that MUG repairs more than 80% of epsilon C residues in vivo. Furthermore, the results show that nucleotide excision repair and recombination are not involved in the processing of epsilon C in E. coli. Based on the mutagenesis data we suggest that epsilon C may be less toxic and less mutagenic than expected. The increased spontaneous mutation rate for G.C --> A.T transition in the ung mug double mutant as compared to the single ung mutant suggest that MUG may be a back-up repair enzyme to the classic uracil-DNA glycosylase.     

Human 3-methyladenine-DNA glycosylase: effect of sequence context on excision, association with PCNA, and stimulation by AP endonuclease

Human 3-methyladenine-DNA glycosylase (MPG protein) is involved in the base excision repair (BER) pathway responsible mainly for the repair of small DNA base modifications. It initiates BER by recognizing DNA adducts and cleaving the glycosylic bond leaving an abasic site. Here, we explore several of the factors that could influence excision of adducts recognized by MPG, including sequence context, effect of APE1, and interaction with other proteins. To investigate sequence context, we used 13 different 25 bp oligodeoxyribonucleotides containing a unique hypoxanthine residue (Hx) and show that the steady-state specificity of Hx excision by MPG varied by 17-fold. If APE1 protein is used in the reaction for Hx removal by MPG, the steady-state kinetic parameters increase by between fivefold and 27-fold, depending on the oligodeoxyribonucleotide. Since MPG has a role in removing adducts such as 3-methyladenine that block DNA synthesis and there is a potential sequence for proliferating cell nuclear antigen (PCNA) interaction, we hypothesized that MPG protein could interact with PCNA, a protein involved in repair and replication. We demonstrate that PCNA associates with MPG using immunoprecipitation with either purified proteins or whole cell extracts. Moreover, PCNA binds to both APE1 and MPG at different sites, and loading PCNA onto a nicked, closed circular substrate with a unique Hx residue enhances MPG catalyzed excision. These data are consistent with an interaction that facilitates repair by MPG or APE1 by association with PCNA. Thus, PCNA could have a role in short-patch BER as well as in long-patch BER. Overall, the data reported here show how multiple factors contribute to the activity of MPG in cells.     

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

Interstrand DNA–DNA cross-links are highly toxic lesions that are important in medicinal chemistry, toxicology, and endogenous biology. In current models of replication-dependent repair, stalling of a replication fork activates the Fanconi anemia pathway and cross-links are “unhooked” by the action of structure-specific endonucleases such as XPF-ERCC1 that make incisions flanking the cross-link. This process generates a double-strand break, which must be subsequently repaired by homologous recombination. Recent work provided evidence for a new, incision-independent unhooking mechanism involving intrusion of a base excision repair (BER) enzyme, NEIL3, into the world of cross-link repair. The evidence suggests that the glycosylase action of NEIL3 unhooks interstrand cross-links derived from an abasic site or the psoralen derivative trioxsalen. If the incision-independent NEIL3 pathway is blocked, repair reverts to the incision-dependent route. In light of the new model invoking participation of NEIL3 in cross-link repair, we consider the possibility that various BER glycosylases or other DNA-processing enzymes might participate in the unhooking of chemically diverse interstrand DNA cross-links.