Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the base excision repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of Escherichia coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity with the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5'2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the "zincless finger" motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNei1. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates.     

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.     

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.     

Plant and fungal Fpg homologs are formamidopyrimidine DNA glycosylases but not 8-oxoguanine DNA glycosylases

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k_obs of over 2500 min^?1.     

Structural analysis of an Escherichia coli endonuclease VIII covalent reaction intermediate

Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential beta-elimination steps. However, Nei differs significantly from Fpg in substrate specificity. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A resolution. The crosslink is derived from a Schiff base intermediate that precedes beta-elimination and is stabilized by reduction with NaBH(4). Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identified. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.     

A novel human DNA glycosylase that removes oxidative DNA damage and is homologous to Escherichia coli endonuclease VIII

Prokaryotes and lower eukaryotes possess redundant activities that remove the plethora of oxidative DNA base damages produced during normal oxidative metabolism and which have been associated with cancer and aging. Thus far, only one oxidized pyrimidine-specific DNA glycosylase has been identified in humans, hNthl. Here, we report the identification of three new putative human DNA glycosylases that are phylogenetically members of the Fpg/Nei family primarily found in the bacterial kingdom. We have characterized one of these, hNEI1, and show it to be functionally homologous to bacterial Nei, that is, its principal substrates are oxidized pyrimidines, it undergoes a lyase reaction by, beta,delta-elimination and traps a Schiff base with a substrate containing thymine glycol (Tg). Furthermore, inactivation of active site residues shown to be important in Escherichia coli Nei inactivate the human enzyme. The hNEI1 gene is located on the long arm of chromosome 15 that is frequently deleted in human cancers.     

Neil3 and NEIL1 DNA Glycosylases Remove Oxidative Damages from Quadruplex DNA and Exhibit Preferences for Lesions in the Telomeric Sequence Context

The telomeric DNA of vertebrates consists of d(TTAGGG)_n tandem repeats, which can form quadruplex DNA structures in vitro and likely in vivo. Despite the fact that the G-rich telomeric DNA is susceptible to oxidation, few biochemical studies of base excision repair in telomeric DNA and quadruplex structures have been done. Here, we show that telomeric DNA containing thymine glycol (Tg), 8-oxo-7,8-dihydroguanine (8-oxoG), guanidinohydantoin (Gh), or spiroiminodihydantoin (Sp) can form quadruplex DNA structures in vitro. We have tested the base excision activities of five mammalian DNA glycosylases (NEIL1, NEIL2, mNeil3, NTH1, and OGG1) on these lesion-containing quadruplex substrates and found that only mNeil3 had excision activity on Tg in quadruplex DNA and that the glycosylase exhibited a strong preference for Tg in the telomeric sequence context. Although Sp and Gh in quadruplex DNA were good substrates for mNeil3 and NEIL1, none of the glycosylases had activity on quadruplex DNA containing 8-oxoG. In addition, NEIL1 but not mNeil3 showed enhanced glycosylase activity on Gh in the telomeric sequence context. These data suggest that one role for Neil3 and NEIL1 is to repair DNA base damages in telomeres in vivo and that Neil3 and Neil1 may function in quadruplex-mediated cellular events, such as gene regulation via removal of damaged bases from quadruplex DNA.     

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 Mg²⁺ 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 Mg²⁺. 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.     

Structural Characterization of a Viral NEIL1 Ortholog Unliganded and Bound to Abasic Site-containing DNA

Endonuclease VIII (Nei) is a DNA glycosylase of the base excision repair pathway that recognizes and excises oxidized pyrimidines. We determined the crystal structures of a NEIL1 ortholog from the giant Mimivirus (MvNei1) unliganded and bound to DNA containing tetrahydrofuran (THF), which is the first structure of any Nei with an abasic site analog. The MvNei1 structures exhibit the same overall architecture as other enzymes of the Fpg/Nei family, which consists of two globular domains joined by a linker region. MvNei1 harbors a zincless finger, first described in human NEIL1, rather than the signature zinc finger generally found in the Fpg/Nei family. In contrast to Escherichia coli Nei, where a dramatic conformational change was observed upon binding DNA, the structure of MvNei1 bound to DNA does not reveal any substantial movement compared with the unliganded enzyme. A protein segment encompassing residues 217–245 in MvNei1 corresponds to the “missing loop” in E. coli Nei and the “αF–β10 loop” in E. coli Fpg, which has been reported to be involved in lesion recognition. Interestingly, the corresponding loop in MvNei1 is ordered in both the unliganded and furan-bound structures, unlike other Fpg/Nei enzymes where the loop is generally ordered in the unliganded enzyme or in complexes with a lesion, and disordered otherwise. In the MvNei1·tetrahydrofuran complex a tyrosine located at the tip of the putative lesion recognition loop stacks against the furan ring; the tyrosine is predicted to adopt a different conformation to accommodate a modified base.All organisms must cope with the generation of potentially lethal or mutagenic oxidative DNA base damage produced by endogenous free radicals. The enzymes that recognize and initiate the repair of these lesions are the DNA glycosylases, which are found ubiquitously in all three kingdoms of life (for reviews see Refs. 1–4). Some of these enzymes are bifunctional, i.e. they catalyze the hydrolysis of the N-glycosyl bond linking a base to a deoxyribose (glycosylase activity) and subsequently cleave the DNA 3′ to the apurinic/apyrimidinic site (lyase activity), whereas others are monofunctional and only carry out the glycosylase reaction, generating abasic sites as products. Structural studies indicate that the DNA glycosylases that recognize oxidative DNA damages fall into two family groups: the helix-hairpin-helix superfamily and the Fpg/Nei family (for reviews see Refs. 1, 5, 6). The helix-hairpin-helix superfamily includes a diverse group of enzymes with varying substrate specificities which nonetheless share a helix-hairpin-helix motif that consists of two α-helices connected by a hairpin loop, followed by a Gly/Pro-rich loop and a conserved catalytic aspartate residue (7). Glycosylase members of the second family, the Fpg/Nei family, share a two-domain architecture: The N-terminal domain consists of a two-layered β-sandwich with two α-helices, whereas the C-terminal domain contains four α-helices, of which two are involved in a conserved helix-two-turn-helix (H2TH)³ motif, and two β-strands that make up the zinc finger motif (2, 6). Both these motifs are involved in DNA binding. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) was the first identified member of this family and was characterized by its ability to recognize and remove formamidopyrimidines (8); however, the principal biological substrate for Fpg is 8-oxoguanine (8-oxoG) (9, 10).More than a decade ago a second DNA glycosylase, which recognizes oxidized pyrimidines, Nei (endonuclease VIII), was identified in E. coli and found to be similar in sequence to Fpg (11, 12). In contrast to Fpg, Nei is only sparsely distributed among prokaryotes (2). Several years ago homologs of this enzyme were identified and characterized in vertebrates, the so-called Nei-like or Neil proteins (13–17). The evolutionary origin of the Neil proteins in vertebrates is unclear (2), and this issue was further confounded several years ago when two Nei-like proteins were identified in the giant Mimivirus (mimicking microbe) (18). This is the largest virus characterized to date with a genomic size of 1.2 Mb, larger than many bacteria (19) and with more than 900 protein coding genes (20). The host for Mimivirus is Acanthamoeba polyphaga, a soil and freshwater protozoan (18).We have recently cloned and characterized the two Nei glycosylases from Mimivirus, MvNei1 (L315) and MvNei2 (L720) (21). Sequence analysis suggests that Mimivirus Nei1 possesses a zincless finger β-hairpin motif first described in human NEIL1, rather than the signature zinc finger (22–24) characteristic of the Fpg/Nei family. Furthermore, we have shown that MvNei1 and MvNei2 have enzymatic properties very similar to their human homologs, NEIL1 and NEIL2 (21). MvNei1 and NEIL1 share substrate preferences for oxidized pyrimidines in duplex DNA. Although MvNei1 and NEIL1 do not recognize 8-oxoguanine, both enzymes cleave its further oxidation products (25, 26), guanidinohydantoin (Gh) and spiroiminodihydantoin, when paired with C (21, 27). Single stranded DNA with the same base lesions, as well as bubble structures, are also substrates for both enzymes (15, 21, 28).Here we report the crystal structures of MvNei1 unliganded and in complex with DNA containing tetrahydrofuran (THF), a structural analog of the cyclic hemiacetal form of an abasic site. Human NEIL1 and MvNei1 bind DNA containing THF but do not cleave the DNA backbone. The MvNei1·THF complex is the first structure of an Nei with an abasic site analog. MvNei1 shares significant structural similarity with human NEIL1 (29). In contrast to E. coli Nei where a dramatic conformational change was observed upon binding DNA (30), the structure of MvNei1 bound to furan containing DNA does not reveal any substantial conformational change compared with the unliganded protein. The MvNei1 loop corresponding to the αF-β9/10 lesion recognition loop of Fpg is ordered in both the unliganded and furan-bound structures, unlike what was reported for other Fpg/Nei enzymes where the loop is generally ordered when the enzyme is unliganded or bound to a lesion, and disordered otherwise. In the MvNei1·THF complex a tyrosine located at the tip of the putative lesion recognition loop stacks against the furan ring; the tyrosine and/or the tip of the loop are predicted to adopt a different conformation in the presence of a modified base.     

The oxidative DNA glycosylases of Mycobacterium tuberculosis exhibit different substrate preferences from their Escherichia coli counterparts

The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs.     

Identification of the Archaeoglobus fulgidus endonuclease III DNA interaction surface using heteronuclear NMR methods.

BACKGROUND: Endonuclease III is the prototype for a family of DNA-repair enzymes that recognize and remove damaged and mismatched bases from DNA via cleavage of the N-glycosidic bond. Crystal structures for endonuclease III, which removes damaged pyrimidines, and MutY, which removes mismatched adenines, show a highly conserved structure. Although there are several models for DNA binding by this family of enzymes, no experimental structures with bound DNA exist for any member of the family. RESULTS: Nuclear magnetic resonance (NMR) spectroscopy chemical-shift perturbation of backbone nuclei (1H, 15N, 13CO) has been used to map the DNA-binding site on Archaeoglobus fulgidus endonuclease III. The experimentally determined interaction surface includes five structural elements: the helix-hairpin-helix (HhH) motif, the iron-sulfur cluster loop (FCL) motif, the pseudo helix-hairpin-helix motif, the helix B-helix C loop, and helix H. The elements form a continuous surface that spans the active site of the enzyme. CONCLUSIONS: The enzyme-DNA interaction surface for endonuclease III contains five elements of the protein structure and suggests that DNA damage recognition may require several specific interactions between the enzyme and the DNA substrate. Because the target DNA used in this study contained a generic apurinic/apyrimidinic (AP) site, the binding interactions we observed for A. fulgidus endonuclease III should apply to all members of the endonuclease III family and several interactions could apply to the endonuclease III/AlkA (3-methyladenine DNA glycosylase) superfamily.     

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5′-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIα-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions −1 and −2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.     

DNA Damage Processing by Human 8-Oxoguanine-DNA Glycosylase Mutants with the Occluded Active Site

8-Oxoguanine-DNA glycosylase (OGG1) removes premutagenic lesion 8-oxoguanine (8-oxo-G) from DNA and then nicks the nascent abasic (apurinic/apyrimidinic) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of 8-oxo-G recognition are not well understood. To comprehend the mechanisms of substrate recognition and processing, we have constructed OGG1 mutants with the active site occluded by replacement of Cys-253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants were characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild type and the mutant OGG1 substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of 8-oxo-G and apurinic/apyrimidinic sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.     

NEIL1 is the major DNA glycosylase that processes 5-hydroxyuracil in the proximity of a DNA single-strand break

5-Hydroxyuracil (5-OHU) in DNA, arising during endogenous DNA damage and caused by ionizing radiation, is removed by the base excision repair pathway. However, in addition to base lesions, ionizing radiation also generates DNA single-strand breaks (SSBs). When these DNA lesions are located in the proximity of each other, this may result in a profound effect on both repair of the damaged base and the SSB. We therefore examined the repair of DNA substrates containing 5-OHU lesions in the proximity of the 3'-end of a SSB. We found that SSB repair by DNA ligase IIIalpha and DNA polymerase beta is impaired by the presence of the nearby 5-OHU lesion, indicating the requirement for a DNA glycosylase which would be able to remove 5-OHU before SSB repair. Subsequently, we found that although both SMUG1 and NEIL1 are able to excise 5-OHU lesions located in the proximity of the 3'-end of a DNA SSB, NEIL1 is more efficient in the repair of these DNA lesions.     

Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1

DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 microM concentration. The measured K(i) was 4.44 +/- 0.15 microM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC(50) was only 343.3 +/- 58.6 and 350 +/- 24.4 microM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 microM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC(50) = 1 microM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC(50) = 76.5 microM, 96 microM, and 187.5 microM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the beta-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.     

Evidence for Base Excision Repair of Oxidative DNA Damage in Chloroplasts of Arabidopsis thaliana

Chloroplasts are the sites of photosynthesis in plants, and they contain their own multicopy, requisite genome. Chloroplasts are also major sites for production of reactive oxygen species, which can damage essential components of the chloroplast, including the chloroplast genome. Compared with mitochondria in animals, relatively little is known about the potential to repair oxidative DNA damage in chloroplasts. Here we provide evidence of DNA glycosylase-lyase/endonuclease activity involved in base excision repair of oxidized pyrimidines in chloroplast protein extracts of Arabidopsis thaliana. Three base excision repair components (two endonuclease III homologs and an apurinic/apyrimidinic endonuclease) that might account for this activity were identified by bioinformatics. Transient expression of protein-green fluorescent protein fusions showed that all three are targeted to the chloroplast and co-localized with chloroplast DNA in nucleoids. The glycosylase-lyase/endonuclease activity of one of the endonuclease III homologs, AtNTH2, which had not previously been characterized, was confirmed in vitro. T-DNA insertions in each of these genes were identified, and the physiological and biochemical phenotypes of the single, double, and triple mutants were analyzed. This mutant analysis revealed the presence of a third glycosylase activity and potentially another pathway for repair of oxidative DNA damage in chloroplasts.Reactive oxygen species (ROS)² are inevitable by-products of metabolism in all aerobic organisms (1). Plants and algae are especially prone to photo-oxidative stress because of ROS generated during oxygenic photosynthesis. Several types of ROS are generated at various sites in the photosynthetic electron transport chain in chloroplasts, and their production is enhanced by such factors as excess or varying light intensities and extremes of temperature, drought, nutrient deficiencies, and herbicides (2). These ROS can damage many chloroplast constituents, including lipids, proteins, pigments, and the multicopy genome.Plants have evolved numerous mechanisms to deal with photo-oxidative stress, including dissipation of excess light energy, synthesis of antioxidant molecules and scavenging enzymes, and targeted repair (2). DNA repair of oxidized bases, such as thymine glycol (TG) or 8-oxoguanine, can be hypothesized as an important element of chloroplast photoprotection. Although there is considerable overlap in both the types of DNA lesions caused by different insults and the targeting of different DNA repair mechanisms, base excision repair (BER) is considered to be the main repair pathway for oxidative DNA damage, at least in the nucleus and mitochondrion (3, 4).BER repairs single damaged bases (because of oxidation, deamination, alkylation, etc.) in DNA by removing them, breaking the phosphodiester backbone, excising the sugar residue at the abasic site, and filling the gap (reviewed in Refs. 5, 6). BER begins with a DNA glycosylase or glycosylase-lyase. There are many types of glycosylases in any given organism and across taxa, and they are distinguishable by their substrate specificity, whether they are monofunctional (glycosylase activity only) or bifunctional (glycosylase plus apurinic/apyrimidinic (AP) lyase activities; see below), by the phylogenetic family in which they reside, and/or by conserved structural characteristics (reviewed in Refs. 6–8). The glycosylases involved in BER of oxidative DNA damage can be roughly divided into those that target either oxidized purines or oxidized pyrimidines (4, 9). For example, TG is a common type of oxidized pyrimidine, which is removed primarily by endonuclease III (Nth), endonuclease VIII (Nei), or their homologs (10). TG is only poorly mutagenic, but it strongly blocks polymerases, inducing cell cycle arrest and potentially cell death if it is not removed.After an appropriate glycosylase cleaves the N-glycosyl bond attaching a damaged base to deoxyribose, leaving an abasic site, the sugar-phosphate backbone is nicked. Bifunctional glycosylases also have an AP lyase activity that cleaves on the 3′ side of the AP site. However, the site still requires the function of a separate AP endonuclease that cuts on the 5′ side of the AP site to remove the 3′-deoxyribose residue at the nick site (11) before repair can continue. In the case of a monofunctional glycosylase, an AP endonuclease nicks the strand on the 5′ side of the AP site. Escherichia coli has two unrelated AP endonucleases, exonuclease III (Xth) and endonuclease IV (Nfo). In humans Ape1/Ref-1 is an Xth homolog, and in yeast Apn1p is an Nfo homolog (5, 12). Following generation of the AP site and nicking of the backbone, the gap is filled by a polymerase in either a short or long patch and then sealed by a ligase.BER of oxidative DNA lesions such TG has been studied intensively in E. coli, yeast, and mammals, whereas comparatively little is known about BER in plants. For example, only two genes involved in BER of oxidized pyrimidines have been characterized previously in the model plant Arabidopsis thaliana (13, 14), and their localization within the plant cell is unknown. An Nth homolog in Arabidopsis, AtNTH1 (At2g31450), has the expected bifunctional glycosylase-lyase activity in vitro (14). The ARP gene (At2g41460) in Arabidopsis encodes an enzyme with AP endonuclease activity (13).Here we present the results of experiments conducted to address whether there is BER of oxidized pyrimidines in the Arabidopsis chloroplast. Chloroplast protein extracts were assayed for glycosylase-lyase/endonuclease activity. The chloroplast localization of ARP, AtNTH1, and AtNTH2, a second Arabidopsis homolog of Nth, was tested experimentally, and the predicted activity of AtNTH2 was confirmed in vitro. In addition, an analysis of T-DNA insertion mutants affecting each of these three BER genes was performed.     

DNA tandem lesion repair by strand displacement synthesis and nucleotide excision repair

DNA tandem lesions are comprised of two contiguously damaged nucleotides. This subset of clustered lesions is produced by a variety of oxidizing agents, including ionizing radiation. Clustered lesions can inhibit base excision repair (BER). We report the effects of tandem lesions composed of a thymine glycol and a 5'-adjacent 2-deoxyribonolactone (LTg) or tetrahydrofuran abasic site (FTg). Some BER enzymes that act on the respective isolated lesions do not accept the tandem lesion as a substrate. For instance, endonuclease III (Nth) does not excise thymine glycol (Tg) when it is part of either tandem lesion. Similarly, endonuclease IV (Nfo) does not incise L or F when they are in tandem with Tg. Long-patch BER overcomes inhibition by the tandem lesion. DNA polymerase beta (Pol beta) carries out strand displacement synthesis, following APE1 incision of the abasic site. Pol beta activity is enhanced by flap endonuclease (FEN1), which cleaves the resulting flap. The tandem lesion is also incised by the bacterial nucleotide excision repair system UvrABC with almost the same efficiency as an isolated Tg. These data reveal two solutions that DNA repair systems can use to counteract the formation of tandem lesions.     

Structure of the uncomplexed DNA repair enzyme endonuclease VIII indicates significant interdomain flexibility

Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei-DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 A resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 A) and Nei-R252A (2.05 A). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a approximately 50 degrees rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme.