Stimulation of human endonuclease III by Y box-binding protein 1 (DNA-binding protein B). Interaction between a base excision repair enzyme and a transcription factor.

Human endonuclease III (hNth1) is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation. Using duplex 2'-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are also members of the endonuclease III family. This difference in rates contrasts with the equality of rates found in Escherichia coli and Saccharomyces cerevisiae endonuclease III homologs. A yeast two-hybrid screen for potential modulators of hNth1 activity revealed interaction with the damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified as DNA-binding protein B (DbpB). The in vitro addition of His(6)YB-1 to hNth1 increased the rate of DNA glycosylase and AP lyase activity. Analysis revealed that YB-1 affects the steady state equilibrium between the covalent hNth1-AP site Schiff base ES intermediate and the noncovalent ES intermediate containing the AP aldehydic sugar and the epsilon-amino group of the hNth1 active site lysine. This equilibrium may be a checkpoint in modulating hNth1 activity.

Identification, characterization, and purification of DNA glycosylase/AP lyases by reductive crosslinking to 2'-deoxyribooligonucleotides containing specific base lesions.

This paper describes a reductive amination crosslinking protocol that facilitates identification and characterization of a class of DNA repair enzymes, DNA glycosylase/AP lyases, which are involved in base excision repair. This crosslinking technique has been used to identify enzymes in crude extracts and in partially purified enzyme preparations, to isolate proteins for sequencing, and to confirm the reaction mechanism of members of this enzyme family. Chemical reduction of the Schiff's base enzyme-substrate intermediate to a stable amine results in the formation of an irreversible covalent bond between the substrate lesion situated within a 2'-deoxyoligonucleotide and the repair enzyme. This complex can be detected by gel electrophoresis and can also be isolated and analyzed by amino acid sequencing.

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.

Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis.

Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that primes DNA repair synthesis by cleaving the DNA backbone 5' of AP sites. The crystal structures of Endonuclease IV and its AP-DNA complex at 1.02 and 1.55 A resolution reveal how an alpha8beta8 TIM barrel fold can bind dsDNA. Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend the DNA approximately 90 degrees, and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme pocket that excludes undamaged nucleotides. These structures suggest three Zn2+ ions directly participate in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bending by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision repair pathway progression.

Mutation identification DNA analysis system (MIDAS) for detection of known mutations.

We introduce a novel experimental strategy for DNA mutation detection named the Mismatch Identification DNA Analysis System (MIDAS) [1, 2], which has an associated isothermal probe amplification step to increase target DNA detection sensitivity to attomole levels. MIDAS exploits DNA glycosylases to remove the sugar moiety on one strand (the probe strand) at a DNA base pair mismatch. The resulting apyrimidinic/ apurinic (AP) site is cleaved by AP endonucleases/lyases either associated with the DNA glycosylase or externally added to the reaction mixture. MIDAS utilizes 32p- or FITC-labeled oligonucleotides as mutation probes. Generally between 20-50 nucleotides in length, the probe hybridizes to the target sequence at the reaction temperature. Mismatch repair enzymes (MREs) then cut the probe at the point of mismatch. Once the probe is cleaved, the fragments become thermally unstable and fall off the target, thereby allowing another full-length probe to hybridize. This oscillating process amplifies the signal (cleaved probe). Cleavage products can be detected by electrophoretic separation followed by autoradiography, or by laser-induced fluorescence-capillary electrophoresis (LIF-CE) of fluorophore-labeled probes in two minutes using a novel CE matrix. In the present experiments, we employed the mesophilic Escherichia coli enzyme deoxyinosine 3'-endonuclease (Endo V), and a novel thermostable T/G DNA glycosylase, TDG mismatch repair enzyme (TDG-MRE). MIDAS differentiated between a clinical sample BRCA 1 wild-type sequence and a BRCA1 185delAG mutation without the need for polymerase chain reaction (PCR). The combination of MIDAS with LIF-CE should make detection of known point mutations, deletions, and insertions a rapid and cost-effective technique well suited for automation.

A new member of the endonuclease III family of DNA repair enzymes that removes methylated purines from DNA.

DNA is constantly exposed to endogenous andexogenous alkylating agents that can modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2]. Alkylation damage is removed by the action of DNA glycosylases, which initiate the base excision repair pathway and protect the sequence information of the genome [3-5]. We have identified a new class of methylpurine DNA glycosylase, designated MpgII, that is a member of the endonuclease III family of DNA repair enzymes. We expressed and purified MpgII from Thermotoga maritima and found that the enzyme releases both 7-methylguanine and 3-methyladenine from DNA. We cloned the MpgII genes from T. maritima and from Aquifex aeolicus and found that both genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli alkA tagA double mutants, which are deficient in the repair of alkylated bases. Analogous genes are found in other Bacteria and Archaea and appear to be the only genes coding for methylpurine DNA glycosylase activity in these organisms. MpgII is the fifth member of the endonuclease III family of DNA repair enzymes, suggesting that the endonuclease III protein scaffold has been modified during evolution to recognize and repair a variety of DNA damage.

Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase.

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T-G and U-G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a beta-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this beta-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.

Purification and characterization of Thermotoga maritima endonuclease IV, a thermostable apurinic/apyrimidinic endonuclease and 3'-repair diesterase.

An endonuclease IV homolog was identified as the product of a conceptual open reading frame in the genome of the hyperthermophilic bacterium Thermotoga maritima. The T. maritima endonuclease IV gene encodes a 287-amino-acid protein with 32% sequence identity to Escherichia coli endonuclease IV. The gene was cloned, and the expressed protein was purified and shown to have enzymatic activities that are characteristic of the endonuclease IV family of DNA repair enzymes, including apurinic/apyrimidinic endonuclease activity and repair activities on 3'-phosphates, 3'-phosphoglycolates, and 3'-trans-4-hydroxy-2-pentenal-5-phosphates. The T. maritima enzyme exhibits enzyme activity at both low and high temperatures. Circular dichroism spectroscopy indicates that T. maritima endonuclease IV has secondary structure similar to that of E. coli endonuclease IV and that the T. maritima endonuclease IV structure is more stable than E. coli endonuclease IV by almost 20 degrees C, beginning to rapidly denature only at temperatures approaching 90 degrees C. The presence of this enzyme, which is part of the DNA base excision repair pathway, suggests that thermophiles use a mechanism similar to that used by mesophiles to deal with the large number of abasic sites that arise in their chromosomes due to the increased rates of DNA damage at elevated temperatures.

Yield of DNA strand breaks after base oxidation of plasmid DNA.

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.

DNA repair: caretakers of the genome?.

Recent results show that the 8-oxoguanine DNA repair system is functionally conserved in bacteria and mammals. The bacterial system protects the genome from the mutagenic effects of oxidative stress; the role of the mammalian system is expected to be similar and defects in it may increase susceptibility to cancer.

Cloning and expression of the cDNA encoding the human homologue of the DNA repair enzyme, Escherichia coli endonuclease III.

We previously purified a bovine pyrimidine hydrate-thymine glycol DNA glycosylase/AP lyase. The amino acid sequence of tryptic bovine peptides was homologous to Escherichia coli endonuclease III, theoretical proteins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and human 3'-expressed sequence tags (3'-ESTs) (Hilbert, T. P., Boorstein, R. J., Kung, H. C., Bolton, P. H., Xing, D., Cunningham, R. P., Teebor, G. W. (1996) Biochemistry 35, 2505-2511). Now the human 3'-EST was used to isolate the cDNA clone encoding the human enzyme, which, when expressed as a GST-fusion protein, demonstrated thymine glycol-DNA glycosylase activity and, after incubation with NaCNBH3, became irreversibly cross-linked to a thymine glycol-containing oligodeoxynucleotide, a reaction characteristic of DNA glycosylase/AP lyases. Amino acids within the active site, DNA binding domains, and [4Fe-4S] cluster of endonuclease III are conserved in the human enzyme. The gene for the human enzyme was localized to chromosome 16p13.2-.3. Genomic sequences encoding putative endonuclease III homologues are present in bacteria, archeons, and eukaryotes. The ubiquitous distribution of endonuclease III-like proteins suggests that the 5,6-double bond of pyrimidines is subject to oxidation, reduction, and/or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism.

Ultraviolet irradiation produces novel endonuclease III-sensitive cytosine photoproducts at dipyrimidine sites.

Ultraviolet light irradiation of DNA in vitro and in vivo induces cyclobutane dimers, (6-4) pyrimidine-pyrimidone photoproducts and a variety of minor products. Using a defined DNA fragment, we have identified two classes of sites that can be cleaved by Escherichia coli endonuclease III: single cytosines whose heat lability corresponds to that of cytosine hydrates and more heat-stable dipyrimidines containing cytosine. The dipyrimidine products are induced at sites suggestive of (6-4) photoproducts but are not recognized as (6-4) photoproducts by radioimmunoassay. Use of oligonucleotides containing a single cyclobutane thymine dimer, a (6-4) photoproduct or the Dewar photoisomer of the (6-4) photoproduct also indicated that these products are not substrates for endonuclease III. We have therefore identified a minor UV photoproduct that has the same sequence specificity as the two major dipyrimidine photoproducts; it may be a minor isomer, a unique derivative or an oxidative lesion confined to dipyrimidine sites. Its biological significance is not yet known but may be masked by the preponderance of major products at the same sites. Its occurrence at the particular site in dipyrimidine sequences involved in the mutagenic action of UV photoproducts suggests that it may play a role in generating C to T transitions that are common UV-induced mutations.

A common mechanism of action for the N-glycosylase activity of DNA N-glycosylase/AP lyases from E. coli and T4.

Duplex oligonucleotides containing the base lesion analogs, O-methylhydroxylamine- and O-benzylhydroxylamine-modified abasic (AP) sites, were substrates for the DNA N-glycosylases endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. These N-glycosylases are known to have associated AP lyase activities. In contrast, uracil DNA N-glycosylase, a simple N-glycosylase which does not have an associated AP lyase activity, was unable to recognize the modified AP sites. Endonuclease III, formamidopyrimidine DNA N-glycosylase and T4 endonuclease V recognized the base lesion analogs as N-glycosylases generating intermediary AP sites which were subsequently cleaved by the enzyme-associated AP lyase activities. Kinetic measurements showed that O-alkoxyamine-modified AP sites were poorer substrates than the presumed physiological substrates. For endonuclease III, DNA containing O-methylhydroxyl-amine or O-benzylhydroxylamine was recognized at 12 and 9% of the rate of DNA containing thymine glycol, respectively, under subsaturating substrate concentrations (as determined by relative Vmax/K(m)). Similarly, with formamidopyrimidine DNA N-glycosylase and T4 endonuclease V. DNA containing O-methylhydroxylamine or O-benzylhydroxylamine was recognized at 4-9% of the efficiency of DNA containing N7-methyl formamidopyrimidine or pyrimidine cyclobutane dimers, respectively. Based on the known structures of these base lesion analogs and the substrate specificities of the N-glycosylases, a common mechanism of action is proposed for DNA N-glycosylases with an associated AP lyase activity.

DNA repair: how yeast repairs radical damage.

Cloning of the OGG1 gene from Saccharomyces cerevisiae has revealed that DNA glycosylases are not necessarily conserved throughout phylogeny, yet there is a DNA-repair protein superfamily with a wide substrate specificity found from bacteria to man.

Methylperoxyl radicals as intermediates in the damage to DNA irradiated in aqueous dimethyl sulfoxide with gamma rays.

Using agarose gel electrophoresis, we have measured the yields of DNA single-strand breaks (SSBs) for plasmid DNA gamma-irradiated in aerobic aqueous solution. Incubation after irradiation with the base damage repair endonucleases formamidopyrimidine-DNA N-glycosylase (FPG) or endonuclease III (endo III) results in an increase in the yield of SSBs. In the absence of dimethyl sulfoxide (DMSO) during irradiation, this increase is consistent with the yields of known substrates for FPG and endo III as determined by gas chromatography/mass spectrometry. After irradiation in the presence of 1 mol dm-3 DMSO, the increase in the yield of SSBs after enzyme incubation was further enhanced by a factor of about 5 to 7. The magnitude of this effect, the inability of acrylamide or oxygen to suppress it, and its attenuation by N,N,N',N'-tetramethylphenylenediamine (TMPD) or glycerol all suggest that the methylperoxyl radical (derived from DMSO) is involved as an intermediate. Reactions of the methylperoxyl radical (or some other species derived from it) do not result in strand break damage, but are responsible for DNA base damages which are recognized by FPG and endo III.

Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide.

We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination). The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate. Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases. We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3. This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE. Phosphorimaging confirmed irreversible cross linking between enzyme and DNA. Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species. Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III. The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C. elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.

Comparison of the effects of UV irradiation on 5-methyl-substituted and unsubstituted pyrimidines in alternating pyrimidine-purine sequences in DNA.

We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions.

Novel DNA binding motifs in the DNA repair enzyme endonuclease III crystal structure.

The 1.85 A crystal structure of endonuclease III, combined with mutational analysis, suggests the structural basis for the DNA binding and catalytic activity of the enzyme. Helix-hairpin-helix (HhH) and [4Fe-4S] cluster loop (FCL) motifs, which we have named for their secondary structure, bracket the cleft separating the two alpha-helical domains of the enzyme. These two novel DNA binding motifs and the solvent-filled pocket in the cleft between them all lie within a positively charged and sequence-conserved surface region. Lys120 and Asp138, both shown by mutagenesis to be catalytically important, lie at the mouth of this pocket, suggesting that this pocket is part of the active site. The positions of the HhH motif and protruding FCL motif, which contains the DNA binding residue Lys191, can accommodate B-form DNA, with a flipped-out base bound within the active site pocket. The identification of HhH and FCL sequence patterns in other DNA binding proteins suggests that these motifs may be a recurrent structural theme for DNA binding proteins.