DNA damage recognition and repair by 3-methyladenine DNA glycosylase I (TAG)

DNA glycosylases help maintain the genome by excising chemically modified bases from DNA. Escherichia coli 3-methyladenine DNA glycosylase I (TAG) specifically catalyzes the removal of the cytotoxic lesion 3-methyladenine (3mA). The molecular basis for the enzymatic recognition and removal of 3mA from DNA is currently a matter of speculation, in part owing to the lack of a structure of a 3mA-specific glycosylase bound to damaged DNA. Here, high-resolution crystal structures of Salmonella typhi TAG in the unliganded form and in a ternary product complex with abasic DNA and 3mA nucleobase are presented. Despite its structural similarity to the helix-hairpin-helix superfamily of DNA glycosylases, TAG has evolved a modified strategy for engaging damaged DNA. In contrast to other glycosylase-DNA structures, the abasic ribose is not flipped into the TAG active site. This is the first structural demonstration that conformational relaxation must occur in the DNA upon base hydrolysis. Together with mutational studies of TAG enzymatic activity, these data provide a model for the specific recognition and hydrolysis of 3mA from DNA.     

Purification and characterization of 3-methyladenine DNA glycosylase I from Escherichia coli.

We have purified 3-methyladenine DNA glycosylase I from Escherichia coli to apparent physical homogeneity. The enzyme preparation produced a single band of Mr 22,500 upon sodium dodecyl sulphate/polyacrylamide gel electrophoresis in good agreement with the molecular weight deduced from the nucleotide sequence of the tag gene (Steinum, A.-L. and Seeberg, E. (1986) Nucl. Acids Res. 14, 3763-3772). HPLC confirmed that the only detectable alkylation product released from (3H)dimethyl sulphate treated DNA was 3-methyladenine. The DNA glycosylase activity showed a broad pH optimum between 6 and 8.5, and no activity below pH 5 and above pH 10. MgSO4, CaCl2 and MnCl2 stimulated enzyme activity, whereas ZnSO4 and FeCl3 inhibited the enzyme at 2 mM concentration. The enzyme was stimulated by caffeine, adenine and 3-methylguanine, and inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and 3-methyladenine. The enzyme showed no detectable endonuclease activity on native, depurinated or alkylated plasmid DNA. However, apurinic sites were introduced in alkylated DNA as judged from the strand breaks formed by mixtures of the tag enzyme and the bacteriophage T4 denV enzyme which has apurinic/apyrimidinic endonuclease activity. It was calculated that wild-type E. coli contains approximately 200 molecules per cell of 3-methyladenine DNA glycosylase I.     

Human cDNA expressing a functional DNA glycosylase excising 3-methyladenine and 7-methylguanine.

A cDNA expression library from a human cell line was introduced into an E. coli strain deficient in the repair of 3-meAde bases in DNA. E. coli strains deficient in the repair of 3-meAde are unusually sensitive to DNA methylating agents. A plasmid pANPG10 (Alkyl-N-Purine-DNA Glycosylase) was rescued from the library based on its ability to reduce the sensitivity of the mutant strain to methylmethane sulfonate. Crude extracts of the E. coli mutant strain hosting the plasmid pANPG10 release both 3-meAde and 7-meGua from DNA. The longest open reading frame in the sequence codes for a polypeptide of 230 amino acids of molecular weight 25.5 kD, with a pI of 9.1. The derived amino acid sequence of the human 3-meAde-DNA glycosylase has 85% sequence identity with the 3-meAde-DNA glycosylase from rat hepatoma cells.     

Excision of 3-methylguanine from alkylated DNA by 3-methyladenine DNA glycosylase I of Escherichia coli.

Escherichia coli has two DNA glycosylases for repair of DNA damage caused by simple alkylating agents. The inducible AlkA DNA glycosylase (3-methyladenine [m3A] DNA glycosylase II) removes several different alkylated bases including m3A and 3-methylguanine (m3G) from DNA, whereas the constitutively expressed Tag enzyme (m3A DNA glycosylase I) has appeared to be specific for excision of m3A. In this communication we have reexamined the substrate specificity of Tag by using synthetic DNA rich in GC base pairs to facilitate detection of any possible methyl-G removal. In such DNA alkylated with [3H]dimethyl sulphate, we found that m3G was excised from double-stranded DNA by both glycosylases, although more efficiently by AlkA than by Tag. This was further confirmed using both N-[3H]methyl-N-nitrosourea- and [3H]dimethyl sulphate-treated native DNA, from which Tag excised m3G with an efficiency that was about 70 times lower than for AlkA. These results can explain the previous observation that high levels of Tag expression will suppress the alkylation sensitivity of alkA mutant cells, further implying that m3G is formed in quantity sufficient to represent an important cytotoxic lesion if left unrepaired in cells exposed to alkylating agents.     

A novel zinc snap motif conveys structural stability to 3-methyladenine DNA glycosylase I

The Escherichia coli 3-methyladenine DNA glycosylase I (TAG) is a DNA repair enzyme that excises 3-methyladenine in DNA and is the smallest member of the helix-hairpin-helix (HhH) superfamily of DNA glycosylases. Despite many studies over the last 25 years, there has been no suggestion that TAG was a metalloprotein. However, here we establish by heteronuclear NMR and other spectroscopic methods that TAG binds 1 eq of Zn2+ extremely tightly. A family of refined NMR structures shows that 4 conserved residues contributed from the amino- and carboxyl-terminal regions of TAG (Cys4, His17, His175, and Cys179) form a Zn2+ binding site. The Zn2+ ion serves to tether the otherwise unstructured amino- and carboxyl-terminal regions of TAG. We propose that this unexpected "zinc snap" motif in the TAG family (CX(12-17)HX(approximately 150)HX(3)C) serves to stabilize the HhH domain thereby mimicking the functional role of protein-protein interactions in larger HhH superfamily members.     

Physical and functional interactions between Escherichia coli MutY glycosylase and mismatch repair protein MutS

Escherichia coli MutY and MutS increase replication fidelity by removing adenines that were misincorporated opposite 7,8-dihydro-8-oxo-deoxyguanines (8-oxoG), G, or C. MutY DNA glycosylase removes adenines from these mismatches through a short-patch base excision repair pathway and thus prevents G:C-to-T:A and A:T-to-G:C mutations. MutS binds to the mismatches and initiates the long-patch mismatch repair on daughter DNA strands. We have previously reported that the human MutY homolog (hMYH) physically and functionally interacts with the human MutS homolog, hMutSalpha (Y. Gu et al., J. Biol. Chem. 277:11135-11142, 2002). Here, we show that a similar relationship between MutY and MutS exists in E. coli. The interaction of MutY and MutS involves the Fe-S domain of MutY and the ATPase domain of MutS. MutS, in eightfold molar excess over MutY, can enhance the binding activity of MutY with an A/8-oxoG mismatch by eightfold. The MutY expression level and activity in mutS mutant strains are sixfold and twofold greater, respectively, than those for the wild-type cells. The frequency of A:T-to-G:C mutations is reduced by two- to threefold in a mutS mutY mutant compared to a mutS mutant. Our results suggest that MutY base excision repair and mismatch repair defend against the mutagenic effect of 8-oxoG lesions in a cooperative manner.     

Crystal structures of 3-methyladenine DNA glycosylase MagIII and the recognition of alkylated bases

DNA glycosylases catalyze the excision of chemically modified bases from DNA. Although most glycosylases are specific to a particular base, the 3-methyladenine (m3A) DNA glycosylases include both highly specific enzymes acting on a single modified base, and enzymes with broader specificity for alkylation-damaged DNA. Our structural understanding of these different enzymatic specificities is currently limited to crystal and NMR structures of the unliganded enzymes and complexes with abasic DNA inhibitors. Presented here are high-resolution crystal structures of the m3A DNA glycosylase from Helicobacter pylori (MagIII) in the unliganded form and bound to alkylated bases 3,9-dimethyladenine and 1,N6-ethenoadenine. These are the first structures of a nucleobase bound in the active site of a m3A glycosylase belonging to the helix-hairpin-helix superfamily. MagIII achieves its specificity for positively-charged m3A not by direct interactions with purine or methyl substituent atoms, but rather by stacking the base between two aromatic side chains in a pocket that excludes 7-methylguanine. We report base excision and DNA binding activities of MagIII active site mutants, together with a structural comparison of the HhH glycosylases.     

Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase

Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.     

The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site

3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision. In contrast to the highly specific 3-methyladenine DNA glycosylase I (E. coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine. We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro. Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine. These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate. Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs. Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site. The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.     

Synergistic interactions between tumor necrosis factor and inhibitors of DNA topoisomerase I and II

TNF is a pleiotropic cytokine that mediates diverse cellular responses, including cytotoxicity, cytostasis, proliferation, differentiation, and the expression of specific genes. Many of these processes require the activity of DNA topoisomerases I and II. We have investigated the interactions of TNF with inhibitors of both topoisomerases in 16-h assays using the murine L929 and human ME-180 cell lines, which undergo a cytotoxic TNF response. Camptothecin, a specific inhibitor of topoisomerase I, enhanced TNF cytotoxicity 150-fold against both cell lines. The topoisomerase II inhibitors VM-26 and VP-16, which stabilize covalent DNA-topoisomerase intermediates, greatly enhance TNF cytotoxicity against both cell lines. The most effective, VM-26, can lower the TNF LD50 to femtomolar levels. In contrast, the topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in ME-180 cells. The large changes in TNF sensitivity induced by drug concentrations that by themselves show no effect, and the opposing synergistic effects of inhibitors with different inhibitory mechanisms (in L929 cells), suggest the active involvement of topoisomerases in TNF-mediated cytotoxicity. The correlation of cytotoxic synergy with the stabilization of DNA strand breaks indicates that DNA damage may play a significant role in TNF-mediated cytotoxicity.     

Physical and functional interactions between uracil-DNA glycosylase and proliferating cell nuclear antigen from the euryarchaeon Pyrococcus furiosus

Uracil-DNA glycosylase (UDG) is an important repair enzyme in all organisms to remove uracil bases from DNA. Recent biochemical studies have revealed that human nuclear UDG (UNG2) forms a multiprotein complex in replication foci and initiates the base excision repair pathway by interacting with proliferating cell nuclear antigen (PCNA). Here, we show the physical and functional interactions between UDG and PCNA from the hyperthermophilic euryarchaeon, Pyrococcus furiosus. The physical interaction between the two proteins was identified by a surface plasmon resonance analysis. Furthermore, the uracil glycosylase activity of P. furiosus UDG is stimulated by P. furiosus PCNA (PfuPCNA) in vitro. This stimulatory effect was observed only when wild type PfuPCNA, but not a monomeric PCNA mutant, was present in the reaction. Mutational analyses revealed that our predicted PCNA-binding region (AKTLF) in P. furiosus UDG is actually important for the interaction with PfuPCNA. This is the first report describing the functional interaction between archaeal UDG and PCNA.     

Physical and functional interaction between human oxidized base-specific DNA glycosylase NEIL1 and flap endonuclease 1

The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.     

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells

3-Methyladenine (3MeA) DNA glycosylases initiate base excision repair by removing 3MeA. These glycosylases also remove a broad spectrum of spontaneous and environmentally induced base lesions in vitro. Mouse cells lacking the Aag 3MeA DNA glycosylase (also known as the Mpg, APNG or ANPG DNA glycosylase) are susceptible to 3MeA-induced S phase arrest, chromosome aberrations and apoptosis, but it is not known if Aag is solely responsible for repair of 3MeA in vivo. Here we show that in Aag^–/– cells, 3MeA lesions disappear from the genome slightly faster than would be expected by spontaneous depurination alone, suggesting that there may be residual repair of 3MeA. However, repair of 3MeA is at least 10 times slower in Aag^–/– cells than in Aag^+/+ cells. Consequently, 24 h after exposure to [³H]MNU, 30% of the original 3MeA burden is intact in Aag^–/– cells, while 3MeA is undetectable in Aag^+/+ cells. Thus, Aag is the major DNA glycosylase for 3MeA repair. We also investigated the in vivo repair kinetics of another Aag substrate, 7-methylguanine. Surprisingly, 7-methylguanine is removed equally efficiently in Aag^+/+ and Aag^–/– cells, suggesting that another DNA glycosylase acts on lesions previously thought to be repaired by Aag.     

Solution structure and base perturbation studies reveal a novel mode of alkylated base recognition by 3-methyladenine DNA glycosylase I

The specific recognition mechanisms of DNA repair glycosylases that remove cationic alkylpurine bases in DNA are not well understood partly due to the absence of structures of these enzymes with their cognate bases. Here we report the solution structure of 3-methyladenine DNA glycosylase I (TAG) in complex with its 3-methyladenine (3-MeA) cognate base, and we have used chemical perturbation of the base in combination with mutagenesis of the enzyme to evaluate the role of hydrogen bonding and pi-cation interactions in alkylated base recognition by this DNA repair enzyme. We find that TAG uses hydrogen bonding with heteroatoms on the base, van der Waals interactions with the 3-Me group, and conventional pi-pi stacking with a conserved Trp side chain to selectively bind neutral 3-MeA over the cationic form of the base. Discrimination against binding of the normal base adenine is derived from direct sensing of the 3-methyl group, leading to an induced-fit conformational change that engulfs the base in a box defined by five aromatic side chains. These findings indicate that base specific recognition by TAG does not involve strong pi-cation interactions, and suggest a novel mechanism for alkylated base recognition and removal.     

Induction of S.cerevisiae MAG 3-methyladenine DNA glycosylase transcript levels in response to DNA damage.

We previously showed that the expression of the Saccharomyces cerevisiae MAG 3-methyladenine (3MeA) DNA glycosylase gene, like that of the E. coli alkA 3MeA DNA glycosylase gene, is induced by alkylating agents. Here we show that the MAG induction mechanism differs from that of alkA, at least in part, because MAG mRNA levels are not only induced by alkylating agents but also by UV light and the UV-mimetic agent 4-nitroquinoline-1-oxide. Unlike some other yeast DNA-damage-inducible genes, MAG expression is not induced by heat shock. The S. cerevisiae MGT1 O6-methylguanine DNA methyltransferase is not involved in regulating MAG gene expression since MAG is efficiently induced in a methyltransferase deficient strain; similarly, MAG glycosylase deficient strains and four other methylmethane sulfonate sensitive strains were normal for alkylation-induced MAG gene expression. However, de novo protein synthesis is required to elevate MAG mRNA levels because MAG induction was abolished in the presence of cycloheximide. MAG mRNA levels were equally well induced in cycling and G1-arrested cells, suggesting that MAG induction is not simply due to a redistribution of cells into a part of the cell cycle which happens to express MAG at high levels, and that the inhibition of DNA synthesis does not act as the inducing signal.     

Protein-nucleotide interactions in E. coli DNA topoisomerase I.

DNA topoisomerases are the enzymes responsible for controlling and maintaining the topological states of DNA. Type IA enzymes work by transiently breaking the phosphodiester backbone of one strand to allow passage of another strand through the break. The protein has to perform complex rearrangements of the DNA, and hence it is likely that different regions of the enzyme bind DNA with different affinities. In order to identify some of the DNA binding sites in the protein, we have solved the structures of several complexes of the 67 kDa N-terminal fragment of Escherichia coli DNA topoisomerase I with mono- and trinucleotides. There are five different binding sites in the complexes, one of which is adjacent to the active site. Two other sites are in the central hole of the protein and may represent general DNA binding regions. The positions of these sites allow us to identify different DNA binding regions and to understand their possible roles in the catalytic cycle.     

Unmasking Anticooperative DNA-binding interactions of vaccinia DNA topoisomerase I

Vaccinia DNA topoisomerase (vTopo) catalyzes highly specific nucleophilic substitution at a single phosphodiester linkage in the pentapyrimidine recognition sequence 5'-(C/T)+5C4+C3+T+2T+1p \N-1 using an active-site tyrosine nucleophile, thereby expelling a 5' hydroxyl leaving group of the DNA. Here, we report the energetic effects of subtle modifications to the major-groove hydrogen-bond donor and acceptor groups of the 3'-GGGAA-5' consensus sequence of the nonscissile strand in the context of duplexes in which the scissile strand length was progressively shortened. We find that the major-groove substitutions become energetically more damaging as the scissile strand is shortened from 32 to 24 and 18 nucleotides, indicating that enzyme interactions with the duplex region present in the 32-mer but not the 24- or 18-mer weaken specific interactions with the DNA major groove. Regardless of strand length, the destabilizing effects of the major-groove substitutions increase as the reaction proceeds from the Michaelis complex to the transition state for DNA cleavage and, finally, to the phosphotyrosine-DNA covalent complex. These length-dependent anticooperative interactions involving the DNA major groove and duplex regions 3' to the cleavage site indicate that the major-groove binding energy is fully realized late during the reaction for full-length substrates but that smaller more flexible duplex substrates feel these interactions earlier along the reaction coordinate. Such anticooperative binding interactions may play a role in strand exchange and supercoil unwinding activities of the enzyme.     

The S. cerevisiae Mag1 3-methyladenine DNA glycosylase modulates susceptibility to homologous recombination

DNA glycosylases, such as the Mag1 3-methyladenine (3MeA) DNA glycosylase, initiate the base excision repair (BER) pathway by removing damaged bases to create abasic apurinic/apyrimidinic (AP) sites that are subsequently repaired by downstream BER enzymes. Although unrepaired base damage may be mutagenic or recombinogenic, BER intermediates (e.g. AP sites and strand breaks) may also be problematic. To investigate the molecular basis for methylation-induced homologous recombination events in Saccharomyces cerevisiae, spontaneous and methylation-induced recombination were studied in strains with varied MAG1 expression levels. We show that cells lacking Mag1 have increased susceptibility to methylation-induced recombination, and that disruption of nucleotide excision repair (NER; rad4) in mag1 cells increases cellular susceptibility to these events. Furthermore, expression of Escherichia coli Tag 3MeA DNA glycosylase suppresses recombination events, providing strong evidence that unrepaired 3MeA lesions induce recombination. Disruption of REV3 (required for polymerase zeta (Pol zeta)) in mag1 rad4 cells causes increased susceptibility to methylation-induced toxicity and recombination, suggesting that Pol zeta can replicate past 3MeAs. However, at subtoxic levels of methylation damage, disruption of REV3 suppresses methylation-induced recombination, indicating that the effects of Pol zeta on recombination are highly dose-dependent. We also show that overproduction of Mag1 can increase the levels of spontaneous recombination, presumably due to increased levels of BER intermediates. However, additional APN1 endonuclease expression or disruption of REV3 does not affect MAG1-induced recombination, suggesting that downstream BER intermediates (e.g. single strand breaks) are responsible for MAG1-induced recombination, rather than uncleaved AP sites. Thus, too little Mag1 sensitizes cells to methylation-induced recombination, while too much Mag1 can put cells at risk of recombination induced by single strand breaks formed during BER.