A discontinuous DNA glycosylase domain in a family of enzymes that excise 5-methylcytosine

DNA cytosine methylation (5-meC) is a widespread epigenetic mark associated to gene silencing. In plants, DEMETER-LIKE (DML) proteins typified by Arabidopsis REPRESSOR OF SILENCING 1 (ROS1) initiate active DNA demethylation by catalyzing 5-meC excision. DML proteins belong to the HhH-GPD superfamily, the largest and most functionally diverse group of DNA glycosylases, but the molecular properties that underlie their capacity to specifically recognize and excise 5-meC are largely unknown. We have found that sequence similarity to HhH-GPD enzymes in DML proteins is actually distributed over two non-contiguous segments connected by a predicted disordered region. We used homology-based modeling to locate candidate residues important for ROS1 function in both segments, and tested our predictions by site-specific mutagenesis. We found that amino acids T606 and D611 are essential for ROS1 DNA glycosylase activity, whereas mutations in either of two aromatic residues (F589 and Y1028) reverse the characteristic ROS1 preference for 5-meC over T. We also found evidence suggesting that ROS1 uses Q607 to flip out 5-meC, while the contiguous N608 residue contributes to sequence-context specificity. In addition to providing novel insights into the molecular basis of 5-meC excision, our results reveal that ROS1 and its DML homologs possess a discontinuous catalytic domain that is unprecedented among known DNA glycosylases.     

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC·G, but not for T·G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.     

ROS1 5-methylcytosine DNA glycosylase is a slow-turnover catalyst that initiates DNA demethylation in a distributive fashion

Arabidopsis ROS1 belongs to a family of plant 5-methycytosine DNA glycosylases that initiate DNA demethylation through base excision. ROS1 displays the remarkable capacity to excise 5-meC, and to a lesser extent T, while retaining the ability to discriminate effectively against C and U. We found that replacement of the C5-methyl group by halogen substituents greatly decreased excision of the target base. Furthermore, 5-meC was excised more efficiently from mismatches, whereas excision of T only occurred when mispaired with G. These results suggest that ROS1 specificity arises by a combination of selective recognition at the active site and thermodynamic stability of the target base. We also found that ROS1 is a low-turnover catalyst because it binds tightly to the abasic site left after 5-meC removal. This binding leads to a highly distributive behaviour of the enzyme on DNA substrates containing multiple 5-meC residues, and may help to avoid generation of double-strand breaks during processing of bimethylated CG dinucleotides. We conclude that the biochemical properties of ROS1 are consistent with its proposed role in protecting the plant genome from excess methylation.     

Demethylation initiated by ROS1 glycosylase involves random sliding along DNA

Active DNA demethylation processes play a critical role in shaping methylation patterns, yet our understanding of the mechanisms involved is still fragmented and incomplete. REPRESSOR OF SILENCING 1 (ROS1) is a prototype member of a family of plant 5-methylcytosine DNA glycosylases that initiate active DNA demethylation through a base excision repair pathway. As ROS1 binds DNA non-specifically, we have critically tested the hypothesis that facilitated diffusion along DNA may contribute to target location by the enzyme. We have found that dissociation of ROS1 from DNA is severely restricted when access to both ends is obstructed by tetraloops obstacles. Unblocking any end facilitates protein dissociation, suggesting that random surface sliding is the main route to a specific target site. We also found that removal of the basic N-terminal domain of ROS1 significantly impairs the sliding capacity of the protein. Finally, we show that sliding increases the catalytic efficiency of ROS1 on 5-meC:G pairs, but not on T:G mispairs, thus suggesting that the enzyme achieves recognition and excision of its two substrate bases by different means. A model is proposed to explain how ROS1 finds its potential targets on DNA.     

A search for base analogs to enhance third-strand binding to 'inverted' target base pairs of triplexes in the pyrimidine/parallel motif.

Eight base analogs were tested as third strand residues in otherwise homopyrimidine strands opposite each of the 'direct' (A.T and G.C) and 'inverted' (T.A and C.G) Watson-Crick base pairs, using UV melting profiles to assess triplex stability. The target duplexes contained 20 A.T base pairs and a central test base pair X.Y, while the third strand contained 20 T residues and a central Z test base. Z included 5-bromo-uracil, 5-propynyluracil, 5-propynylcytosine, 5-methyl-cytosine, 5-bromocytosine, hypoxanthine, 2-amino-purine and 2,6-diaminopurine. Some of the base analogs enhanced third strand binding to the target duplex with one or other 'inverted' central base pair relative to the binding afforded by any of the canonical bases. Other analogs did the same for the duplexes with the 'direct' target pairs. The increasing order of triplex stabilization by these base analogs is: opposite the 'inverted' base pairs, for T.A, A < C < 5-pC < 5-pU < T < 5-BrC < 5-meC < 5-BrU < 2-AP < 2,6-DAP < Hy < G, for C.G, 2-AP < A < Hy < G < 5-pC < 5-BrC < 5-meC < C < 2,6-DAP < T < 5-BrU < 5-pU; opposite the 'direct' base pairs, for A.T, 2-AP < A < 5-meC < C < G < Hy < 2,6-DAP < 5-pU < T = 5-BrU < 5-BrC < 5-pC, for G.C, G < 2,6-DAP < 2-AP < A < Hy < T < 5-BrU < 5-pU < 5-pC < 5-BrC < C < 5-meC.     

DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues.

Derivatives of phage M13 were constructed and used for the in vitro preparation of heteroduplex DNA molecules containing base/base mismatches that mimick DNA lesions caused by hydrolytic deamination of 5-meC residues in Escherichia coli DNA (i.e. they carry a T/G mismatch in the special sequence context provided by the recognition site -CCA/TGG-of the Dcm-methyltransferase). Upon introduction of these heteroduplex DNAs into CaCl2-treated E. coli cells, the mismatches are efficiently repaired with high bias in favour of the DNA strand containing the mismatched guanine residue. This special DNA mismatch-repair operates on fully dam-methylated DNA and is independent of gene mutH. It thus fulfills the salient requirements of a repair pathway responsible for counteracting the spontaneous hydrolytic deamination of 5-meC in vivo. The repair efficiency is boosted by a 5-methyl group present on the cytosine residue at the next-nearest position to the 5' side of the mismatched guanine. The repair is severely impaired in host strains carrying a mutation in any of the three loci dcm, mutL and mutS.     

The DNA Repair Protein XRCC1 Functions in the Plant DNA Demethylation Pathway by Stimulating Cytosine Methylation (5-meC) Excision,Gap Tailoring,and DNA Ligation

DNA methylation patterns are the dynamic outcome of antagonist methylation and demethylation mechanisms, but the latter are still poorly understood. Active DNA demethylation in plants is mediated by a family of DNA glycosylases typified by Arabidopsis ROS1 (repressor of silencing 1). ROS1 and its homologs remove 5-methylcytosine and incise the sugar backbone at the abasic site, thus initiating a base excision repair pathway that finally inserts an unmethylated cytosine. The DNA 3′-phosphatase ZDP processes some of the incision products generated by ROS1, allowing subsequent DNA polymerization and ligation steps. In this work, we examined the possible role of plant XRCC1 (x-ray cross-complementing group protein 1) in DNA demethylation. We found that XRCC1 interacts in vitro with ROS1 and ZDP and stimulates the enzymatic activity of both proteins. Furthermore, extracts from xrcc1 mutant plants exhibit a reduced capacity to complete DNA demethylation initiated by ROS1. An anti-XRCC1 antibody inhibits removal of the blocking 3′-phosphate in the single-nucleotide gap generated during demethylation and reduces the capacity of Arabidopsis cell extracts to ligate a nicked DNA intermediate. Our results suggest that XRCC1 is a component of plant base excision repair and functions at several stages during active DNA demethylation in Arabidopsis.     

Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity

DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1(-/-)) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1(-/-) mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype.     

Active DNA demethylation in plants: 20 years of discovery and beyond

Maintaining proper DNA methylation levels in the genome requires active demethylation of DNA. However, removing the methyl group from a modified cytosine is chemically difficult and therefore, the underlying mechanism of demethylation had remained unclear for many years. The discovery of the first eukaryotic DNA demethylase, Arabidopsis thaliana REPRESSOR OF SILENCING 1 (ROS1), led to elucidation of the 5-methylcytosine base excision repair mechanism of active DNA demethylation. In the 20 years since ROS1 was discovered, our understanding of this active DNA demethylation pathway, as well as its regulation and biological functions in plants, has greatly expanded. These exciting developments have laid the groundwork for further dissecting the regulatory mechanisms of active DNA demethylation, with potential applications in epigenome editing to facilitate crop breeding and gene therapy.     

Crystal structure of human methyl-binding domain IV glycosylase bound to abasic DNA

The mammalian repair protein MBD4 (methyl-CpG-binding domain IV) excises thymine from mutagenic G·T mispairs generated by deamination of 5-methylcytosine (mC), and downstream base excision repair proteins restore a G·C pair. MBD4 is also implicated in active DNA demethylation by initiating base excision repair of G·T mispairs generated by a deaminase enzyme. The question of how mismatch glycosylases attain specificity for excising thymine from G·T, but not A·T, pairs remains largely unresolved. Here, we report a crystal structure of the glycosylase domain of human MBD4 (residues 427-580) bound to DNA containing an abasic nucleotide paired with guanine, providing a glimpse of the enzyme-product complex. The mismatched guanine remains intrahelical, nestled into a recognition pocket. MBD4 provides selective interactions with the mismatched guanine (N1H, N2H(2)) that are not compatible with adenine, which likely confer mismatch specificity. The structure reveals no interactions that would be expected to provide the MBD4 glycosylase domain with specificity for acting at CpG sites. Accordingly, we find modest 1.5- to 2.7-fold reductions in G·T activity upon altering the CpG context. In contrast, 37- to 580-fold effects were observed previously for thymine DNA glycosylase. These findings suggest that specificity of MBD4 for acting at CpG sites depends largely on its methyl-CpG-binding domain, which binds preferably to G·T mispairs in a methylated CpG site. MBD4 glycosylase cannot excise 5-formylcytosine (fC) or 5-carboxylcytosine (caC), intermediates in a Tet (ten eleven translocation)-initiated DNA demethylation pathway. Our structure suggests that MBD4 does not provide the electrostatic interactions needed to excise these oxidized forms of mC.     

Counteracting the mutagenic effect of hydrolytic deamination of DNA 5-methylcytosine residues at high temperature: DNA mismatch N-glycosylase Mig.Mth of the thermophilic archaeon Methanobacterium thermoautotrophicum THF.

Spontaneous hydrolytic deamination of DNA 5-methylcytosine residues gives rise to T/G mismatches which are pre-mutagenic lesions requiring DNA repair. For fundamental reasons, the significance of this and other processes lowering genetic fidelity must be accentuated at elevated temperatures, making thermophilic organisms attractive objects for studying how cells cope with thermal noise threatening the integrity of their genetic information. Gene mig of Methanobacterium thermoautotrophicum THF, an anaerobic archaeon with an optimal growth temperature of 65 degrees C, was isolated and its product (Mig.Mth; EC3.2.2-) shown to be a T/G-selective DNA thymine N-glycosylase with the properties required for counteracting the mutagenic effect of hydrolytic 5-meC deamination. The enzyme acts on T/G and U/G oppositions with similar efficiency; G/G, A/G, T/C and U/C are minor substrates; no other opposition of common nucleobases is attacked and no removal of U from single-stranded DNA is observed. Substrate preferences are modulated by sequence context. Together with the results presented here, one example of an enzyme directed against the hydrolytic deamination damage of 5-meC is known from each of the three phylogenetic kingdoms; entry into the repair pathway is glycosylytic in the eukaryotic and the archaeal case, whereas the eubacterial repair starts with an endonucleolytic DNA incision.     

Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins

The Escherichia coli AlkB protein repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions in DNA and RNA by oxidative demethylation, a reaction requiring ferrous iron and 2-oxoglutarate as cofactor and co-substrate, respectively. Here, we have studied the activity of AlkB proteins on 3-methylthymine (3-meT) and 1-methylguanine (1-meG), two minor lesions which are structurally analogous to 1-meA and 3-meC. AlkB as well as the human AlkB homologues, hABH2 and hABH3, were all able to demethylate 3-meT in a DNA oligonucleotide containing a single 3-meT residue. Also, 1-meG lesions introduced by chemical methylation of tRNA were efficiently removed by AlkB. Unlike 1-meA and 3-meC, nucleosides or bases corresponding to 1-meG or 3-meT did not stimulate the uncoupled, AlkB-mediated decarboxylation of 2-oxoglutarate. Our data show that 3-meT and 1-meG are repaired by AlkB, but indicate that the recognition of these substrates is different from that in the case of 1-meA and 3-meC.     

AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis

DNA methylation is a primary epigenetic modification regulating gene expression and chromatin structure in many eukaryotes. Plants have a unique DNA demethylation system in that 5-methylcytosine (5mC) is directly removed by DNA demethylases, such as DME/ROS1 family proteins, but little is known about the downstream events. During 5mC excision, DME produces 3′-phosphor-α, β-unsaturated aldehyde and 3′-phosphate by successive β- and δ-eliminations, respectively. The kinetic studies revealed that these 3′-blocking lesions persist for a significant amount of time and at least two different enzyme activities are required to immediately process them. We demonstrate that Arabidopsis AP endonucleases APE1L, APE2 and ARP have distinct functions to process such harmful lesions to allow nucleotide extension. DME expression is toxic to E. coli due to excessive 5mC excision, but expression of APE1L or ARP significantly reduces DME-induced cytotoxicity. Finally, we propose a model of base excision repair and DNA demethylation pathway unique to plants.     

Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites

Thymine DNA glycosylase (TDG) excises T from G·T mispairs and is thought to initiate base excision repair (BER) of deaminated 5-methylcytosine (mC). Recent studies show that TDG, including its glycosylase activity, is essential for active DNA demethylation and embryonic development. These and other findings suggest that active demethylation could involve mC deamination by a deaminase, giving a G·T mispair followed by TDG-initiated BER. An alternative proposal is that demethylation could involve iterative oxidation of mC to 5-hydroxymethylcytosine (hmC) and then to 5-formylcytosine (fC) and 5-carboxylcytosine (caC), mediated by a Tet (ten eleven translocation) enzyme, with conversion of caC to C by a putative decarboxylase. Our previous studies suggest that TDG could excise fC and caC from DNA, which could provide another potential demethylation mechanism. We show here that TDG rapidly removes fC, with higher activity than for G·T mispairs, and has substantial caC excision activity, yet it cannot remove hmC. TDG excision of fC and caC, oxidation products of mC, is consistent with its strong specificity for excising bases from a CpG context. Our findings reveal a remarkable new aspect of specificity for TDG, inform its catalytic mechanism, and suggest that TDG could protect against fC-induced mutagenesis. The results also suggest a new potential mechanism for active DNA demethylation, involving TDG excision of Tet-produced fC (or caC) and subsequent BER. Such a mechanism obviates the need for a decarboxylase and is consistent with findings that TDG glycosylase activity is essential for active demethylation and embryonic development, as are mechanisms involving TDG excision of deaminated mC or hmC.     

Substrate specificities of bacterial and human AlkB proteins

Methylating agents introduce cytotoxic 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) residues into nucleic acids, and it was recently demonstrated that the Escherichia coli AlkB protein and two human homologues, hABH2 and hABH3, can remove these lesions from DNA by oxidative demethylation. Moreover, AlkB and hABH3 were also found to remove 1-meA and 3-meC from RNA, suggesting that cellular RNA repair can occur. We have here studied the preference of AlkB, hABH2 and hABH3 for single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), and show that AlkB and hABH3 prefer ssDNA, while hABH2 prefers dsDNA. This was consistently observed with three different oligonucleotide substrates, implying that the specificity for single-stranded versus double-stranded DNA is sequence independent. The dsDNA preference of hABH2 was observed only in the presence of magnesium. The activity of the enzymes on single-stranded RNA (ssRNA), double-stranded RNA (dsRNA) and DNA/RNA hybrids was also investigated, and the results generally confirm the notion that while AlkB and hABH3 tend to prefer single-stranded nucleic acids, hABH2 is more active on double-stranded substrates. These results may contribute to identifying the main substrates of bacterial and human AlkB proteins in vivo.