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. Author manuscript; available in PMC: 2016 Aug 1.

Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites

Alfonso Bellacosaa,*,Alexander C Drohatb,*
aCancer Epigenetics Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia PA 19111, United States
bDepartment of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore MD 21201, United States
*

Corresponding authors: Tel.:+1-215-728-1041; fax: +1- 215-214-1737,Alfonso.Bellacosa@fccc.edu (Alfonso Bellacosa). Tel.: +1-410-706-8118; fax: +1-410-706-8297,adrohat@som.umaryland.edu (Alexander C. Drohat)

Issue date 2015 Aug.

PMCID: PMC4903958  NIHMSID: NIHMS733191  PMID:26021671
The publisher's version of this article is available atDNA Repair (Amst)

Abstract

Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, Methyl Binding Domain 4 (MBD4) and Thymine DNA Glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G·T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.

Keywords: Base excision repair, DNA glycosylase, Deamination, Oxidation, Demethylation, 5-methylcytosine, CpG site, Methyl binding domain 4, Thymine DNA glycosylase, G·T mispair

1. Introduction

The susceptibility of cytosine to a variety of chemical modifications [1] creates an interesting conundrum during evolution: its intrinsic reactivity allows for enzymatic modifications that could be adapted to articulate novel epigenetic functions, but it can also alter the coded genetic information and lead to mutations. For this reason, a discourse on the genetic stability of cytosine must also take into consideration the related and intertwined problem of epigenetic integrity. Thus, enzymatic modifications at the 5′ position of cytosine, generally in the context of CpG dinucleotides, create 5-methylcytosine (5mC), and its progressively oxidized variants, 5-hydroxymethylcytosine (5hmC), 5-formylcytosine(5fC) and 5-carboxylcytosine (5caC); these bases are all important for epigenetic regulation [2]. On the other hand, deamination of cytosine and some of its 5′ derivatives has the potential to create miscoding transition mutations.

There are numerous examples of DNA repair processes closely linked to transcriptional regulation; e.g. nucleotide excision repair factors involved in basal transcription or chromatin remodeling. However, only in the case of CpG sites are genomic and epigenomic stability so jointly rooted in the unique chemistry of cytosine and therefore, unsurprisingly, handled by the same repair system: base excision repair (BER). Research conducted in the past five years is beginning to answer key questions about how this repair system keeps the detrimental consequences of genomic instability of CpG sites under control, while regulating complex epigenetic processes such as DNA demethylation.

2. Two sides of cytosine chemical modifications: regulating gene expression and susceptibility to mutagenic deamination

2.1 Cytosine methylation and oxidation: features and functions

The 5′ position of cytosine is not involved in hydrogen bonding with guanine and can therefore accept many substitutions with minimal to no structural impact to the DNA helix. Cytosine methylation by DNA methyltransferases occurs in bacteria, as a part of restriction-modification systems that limit bacteriophage propagation[3], in plants, as required for development, transcriptional regulation and genome architecture[4], and at low levels in fruit flies, where it is associated with genome stability and silencing of retrotransposons [5].It is with the emergence of vertebrates that DNA methylation becomes present at high levels throughout the genome, and the benefits afforded by a fifth DNA base, 5mC, are fully realized. In mammals, 5mC is present at levels corresponding to 1% of the other bases, found mostly at CpG dinucleotides. Approximately 80–90% of CpG sites are methylated in a temporally and spatially restricted fashion [6–8], whereas the so-called CpG islands found largely in the promoters of housekeeping genes and some tissue-specific genes typically have a high density of non-methylated CpG sites [9].

In mammals, 5mC is extensively used for transcriptional repression, a function derived from its vestigial role in the suppression of parasitic sequences (transposable elements, proviral DNA, and transgenes). DNA methylation is associated with silenced genes and reinforces repressive chromatin either by directly inhibiting the binding of transactivating transcription factors to promoters and regulatory sequences, or by attracting proteins containing domains that recognize methylated DNA, such as Methyl-CpG binding domain (MBD) proteins, among others, which in turn recruit a repressive complex[10,11].Related to transcriptional silencing are two other functions of DNA methylation, X-chromosome in activation and imprinting [12–16].

Importantly, cytosine methylation is deranged in cancer. CpG islands and other typically nonmethylated regions can become hypermethylated, silencing many genes including tumor suppressors[17–19]. Similarly, loss of imprinting, i.e. loss of the allelic methylation differences at imprinted genes, has also been described in cancer[20].In addition, methylation at some CpG islands may occur during aging[21].

Cytosine methylation is mediated by DNA methyltransferases(DNMTs). The maintenance enzyme, DNMT1, converts hemimethylated CpG sites to fully methylated ones, as needed to maintain the epigenetic mark following DNA replication[22]. Thede novo DNA methyltransferases, DNMT3a and DNMT3b, prefer unmethylated DNA substrates and mediate the original placing of methylation marks during early development[23], but also contribute to the maintenance of methylation in adult tissues [24].

Whether the methylation mark could only be removed passively, by dilution through successive cell divisions, or also actively, via direct enzymatic activity, was under debate until a few years ago, when the molecular basis of active DNA demethylation began to emerge. The detailed pathways and biochemistry of active DNA demethylation are still being elucidated, but the overall features are relatively well defined and involve multi-step oxidation of 5mC by newly discovered enzymes followed by novel activities of known DNA base excision repair glycosylases.

Oxidation of 5mC is catalyzed by Fe2+- and α-ketoglutarate-dependent dioxygenases of the Ten Eleven Translocation (TET) family. The family includes the prototypical and eponymous TET1, originally identified as a Mixed Lineage Leukemia (MLL) fusion partner in acute myelogenous leukemia characterized by the t(10;11) translocation[25,26], and the highly related proteins TET2 and TET3 [27].These enzymes oxidize 5mC to 5hmC[28] and successively to 5fC and 5caC(Fig. 1) [29,30].

Fig. 1.

Fig. 1

Pathway for active DNA demethylation mediated by TET enzymes followed by TDG-initiated BER. TET enzymes (noted in red) catalyze stepwise oxidation of 5mC to 5hmC, 5fC, and 5caC. TDG excises 5fC and 5caC and subsequent BER steps yield cytosine.

Whereas oxidation of 5mC represents an initial step of active demethylation, a full demethylation is achieved when 5fC and 5caC are removed by thymine DNA glycosylase (TDG) and subsequent BER leading to incorporation of cytosine (vide infra)[29,31] (Fig. 1). However, it is likely that 5hmC, 5fC and 5caC are not merely intermediates of active demethylation, but also have epigenetic functions on their own. It remains to be determined how TET activity is regulated, i.e. how the oxidation of 5mC stops to 5hmC or proceeds to 5fC and 5caC.

5hmC levels correspond to 5–10% and 40% of 5mC in embryonic stem cells and Purkinje neurons of the cerebellum, respectively [28,32]. 5hmC is very enriched at enhancers of tissue-specific genes and its levels further increase with enhancer activity and differentiation. One possibility is that 5mC oxidation acts as a switch that promotes transcription factor binding; alternatively, it is transcription factor binding that tethers TET proteins onto enhancers promoting an active transcriptional milieu.

The steady-state levels of 5fC and 5caC are much lower than those of 5hmC, corresponding to approximately 0.03% and 0.01% of 5mC levels, respectively [29,30,33]. Interestingly, 5fC and 5caC levels increase upon TDG knockdown, particularly at promoters and enhancers, suggesting that they may be involved in transcriptional activation[34–39]. In other words, since 5mC and 5hmC levels are very low at promoters, it is possible that rapid generation of 5mC and its immediate oxidation are actually associated with promoter activity. More evidence is needed to confirm this model that defies the traditional view of methylation as a silencing, repressive mark; however, rapid cycles of methylation and demethylation have been described at estrogen-regulated promoters during transcriptional activation[40,41]and would be consistent with this model.

2.2 Mutagenesis by deamination of 5mC

The spontaneous decay of DNA bases includes hydrolytic deamination of purines and pyrimidines that contain an exocyclic amino group. While hypoxanthine and xanthine are generated at a relatively slow rate by deamination of adenine and guanine, respectively, deamination of pyrimidines is potentially highly mutagenic, as it occurs at a 50-fold higher rate of approximately 200300 events per cell per day [42–45].Deamination of C and 5mC generates G·U and G·T mismatches, respectively, which, upon DNA replication, cause C to T transition mutations. Notably, for 5mC deamination, these mutations will arise predominantly in the context of CpG sites. Thus, the benefits of a methylated genome come at a price of increased susceptibility to mutations. Uracil was excluded as a component of DNA early in evolution and is efficiently excised by uracil glycosylase activities [44]. However, the fate of 5mCat CpG sites is more complex. First, the deamination rate of 5mC is approximately three times that of C [46]. Second, whereas uracil can be recognized as being foreign to DNA, enzymatic removal of thymine from G·T mispairs has to be balanced against the erroneous and mutagenic excision of thymine from A·T base pairs (vide infra).Similarly, deamination of 5hmC to 5hmU, which base-pairs with adenine, also has the potential to cause transition mutations at CpG sites. Notably, 5hmU can be excised by TDG, MBD4, SMUG1 and NEIL1 [47].During evolution, reiteration of this mutagenic process is believed to have led to under-representation of the CpG dinucleotide in the mammalian genome [48]. Evolutionary consequences are an excess of transition mutations in ortholog genes of humanvs. other primates (interspecies evolution), and elevated single nucleotide polymorphisms at CpG sites among humans (intraspecies genetic variation) [49,50]. Similarly, the accelerated genetic evolution in somatic cells, which characterizes tumorigenesis, also exploits the spontaneous decay of pyrimidines, and it is estimated that nearly one-third of all cancer mutations in coding regions originates by deamination of cytosine and 5mC at CpG sites [51,52].

3. Role of TDG and MBD4 in maintaining the genetic integrity of CpG sites

3.1 Overview – countering the threat posed by 5mC deamination

While the repair of G·T mismatches resulting from DNA replication errors is directed at the new strand via mismatch repair (MMR), repair of 5mC deamination must involve excision of T (not G) from the G·T mispair, and subsequent BER to restore the G·C pair. Two unrelated mammalian enzymes possess this capability: thymine DNA glycosylase (TDG) [53,54] and methyl binding domain 4 (MBD4) [55,56]. Consistent with a role in countering mutations caused by 5mC deamination, MBD4 and TDG have specificity for acting on mispairs arising in a CpG context, though they employ different mechanisms to do so[57].While most DNA glycosylases remove lesions that are clearly foreign to DNA (e.g., uracil, 8-oxoguanine), MBD4 and TDG remove acanonical base from a mismatched pair. As such, these enzymes strike a balance between potentially conflicting needs for efficient removal of T from mutagenic G·T lesions and minimal excision of T from the vast background of A·T pairs, which can be mutagenic and cytotoxic [58,59]. Such a compromise could lead to suboptimal G·T glycosylase activity, as indicated for TDG [60], which in turn could potentially contribute to the high mutational frequency at CpG sites in cancer and genetic disease [61,62]. Structural and biochemical studies have answered some questions about how these enzymes selectively excise T from G·T mispairs, but the detailed mechanism remains largely unknown.

3.2 MBD4-initiated repair of deaminated 5mC

Human MBD4 (580 residues) contains a methyl-CpG binding domain (MBD4MBD; 82–147) and a C-terminal glycosylase domain (426–580) separated by a region of unknown function that appears to be largely disordered (Fig. 2A, B, C) [55,56,63,64]. The glycosylase domain (MBD4GD) retains the catalytic activity of full-length MBD4 [55,56,63]. The MBD binds preferably to fully methylated CpG sites and their deamination derivatives (5mCG/5mCG, 5mCG/TG) [55,64,65], thereby targeting the protein to regions enriched in these sites (Fig. 2B, D). Confirming a biological role for MBD4 in countering mutations caused by 5mC deamination, deficiency of MBD4(Mbd4−/−) in mice causes elevated CT mutations at CpG sites, and accelerates intestinal tumor formation for mice with a predisposing mutation in the adenomatous polyposis coli gene (ApcMin/+) [62,66]. MBD4 is mutated in many cancers that exhibit microsatellite instability (MSI), which can lead to expression of a truncated protein (MBD4tru) that contains the MBD but not the glycosylase domain[67–69]. Notably, ectopic expression of MBD4tru causes a dominant negative impairment of repair in cancer cells and elevated mutations at CpG sites and other locations[67,68]. Recent studies have advanced our understanding of how MBD4 protects against mutations at CpG sites.

Fig. 2.

Fig. 2

Structures of MBD4. (A) Schematic primary structure of MBD4. The MBD and the glycosylase domain are structured, while other regions appear to be disordered. (B) Crystal structure of the MBD bound to a G·T mispair in a CpG context, i.e., 5mCG/TG (PDBID: 3VXV). The DNA is shown as gray lines with surface representation, the G·T mispair is shown in cyan and the flanking C·G pair (CpG context) is in yellow (with N and O atoms blue and red). The same coloring scheme is used in panels C, D, and E. Note that the structures in panels B through E are oriented in the same way with respect to the position of the mismatched guanine (cyan, with an asterisk).(C) Structure of the MBD4 glycosylase domain bound to DNA, with thymine of a G·T mispair flipped into the active site (PDBID: 4E9G). (D) Close up view of the MBD (as in panel B) shows that it recognizes an 5mCG/GT dinucleotide using conserved Arg and other residues together with water molecules. (E) Close-up view of contacts the glycosylase forms with the flipped thymine, via Tyr and Gln side chains and backbone contacts. Also shown are contacts with mismatched G, and the Arg side chain that fills the void created by thymine flipping.

3.2.1 Glycosylase domain of MBD4

Structures of the DNA-free glycosylase domain (MBD4GD) confirmed that it belongs to the Endo III or helix-hairpin-helix (HhH) superfamily of repair enzymes [47,63,70]. MBD4GD is most similar (structurally) to two HhH enzymes that recognize mismatched pairs, the 8oxoG·A mismatch glycosylase MutY [71,72] and the thermophilic G·T mismatch glycosylase MIG [73]. However, MBD4GD (~155 residues) is smaller than MutY (352 residues) and MIG (221 residues) and it lacks the iron-sulfur cluster [4Fe4S] domain found in these other enzymes.

The first structure of DNA-bound MBD4GD provided a snapshot of the enzyme-product (E·P) complex, with a tetrahydrofuran (THF) a basic-site analog flipped into the active site [74]. Structures were also reported for lesion recognition (enzyme-substrate) complexes, with a catalytically inactive form of MBD4GD bound to a G·T or a G·5hmU mispair (Fig. 2C, E) [75,76]. These structures revealed that the mismatched guanine remains in the DNA helix and is contacted at N1H and N2H2 by backbone oxygens of the enzyme (Fig. 2C, E). These contacts are not compatible with adenine and may account for some degree of the stringent specificity of MBD4 for G·T mispairs relative to A·T pairs [56,77].However, the role of these selective contacts has not been directly examined, and mismatch specificity may also depend on the nature of G·T mispairs, which adopt a wobble structure and are less stable (thermodynamically) and more dynamic than A·T pairs[78–80].

Remarkably, MBD4GD does not contact the G·C pair immediately 3′ of the flipped nucleotide, indicating that the glycosylase domain lacks specificity for acting on lesions in a CpG context. This accounts for findings that MBD4GD activity does not depend on the methylation status of a CpG site that harbors a target lesion [55,56,77] and that activity for a G·T mispair is not dramatically altered when the flanking G·C pair(CpG context) is replaced by one of the other three base pairs[74]. By contrast, the G·T activity of TDG depends strongly on the presence of a guanine on the 3′ side of the target thymine[57,81,82]. As discussed below, specificity for CpG sites resides in the MBD of MBD4.

The structures of lesion-recognition (ES) complexes reveal that the enzyme contacts O2 and O4 of the flipped base using a Try hydroxyl and a backbone amide NH, respectively(Fig. 2E)[75,76]. MBD4GD also uses a Gln side chain to contact N3H and O4 of the flipped T, 5hmU, or other thymine analogue. Notably, these Gln contacts would not be compatible with cytosine or its C5-substituted analogues (5mC, 5hmC, 5fC, 5caC). Although rotation of the Gln side chain could potentially allow favorable contacts with cytosines, its orientation is stabilized by a hydrogen bond to the backbone (Fig. 2E). These observations may account, in part, for the inability of MBD4 to excise cytosine analogues. Another potential factor is the much greater stability of guanine base pairs with cytosine analogues (G·5xC) as compared to guanine mispairs with thymine and its analogues (G·T, G·5xU).Lack of MBD4 activity for oxidized forms of 5mC is not likely explained by steric effects, because the enzyme tolerates bulky substituents at the C5 position (Fig. 2E) [74,77].

3.2.2 Targeting by the methyl-CpG-methyl-binding domain of MBD4

MBD4 localizes to methylated foci when it is ectopically expressed in mammalian cells, a function that is attributable to its methyl-CpG binding domain (MBD4MBD)[55,64,65]. MBD4 binds more tightly to fully methylated CpG sites (5mCG/5mCG) than to hemi- or non-methylated sites, similar to other MBD proteins (MBD1, MBD2, MeCP2). Unlike these others, MBD4 also binds methylated sites that have undergone deamination, to give a G·T mispair (5mCG/TG) [55,65], or oxidation by a Tet enzyme to give 5hmC(5mCG/5hmCG) [65]. MBD4MBD binds the major groove and employs a conserved Arg and other side chains, together with a network of water molecules to recognize these sites (Fig. 2D)[65].

Observations that MBDs bind the major grove of DNA and glycosylases bind the minor groove prompted the idea that targeting of MBD4 might involve simultaneous binding of the MBD and the glycosylase domain to a single damaged site [73]. However, as noted previously [63], and shown inFigures 2B and 2C, the sharp (57°) bend in DNA imposed by the glycosylase domain[74–76]seems likely to disrupt binding of the MBD, which binds straight DNA [65]. Taken together, findings to date indicate that the two domains bind unique sites of DNA, and that the MBD targets full-length MBD4 to regions of methylated CpG sites so that the glycosylase domain can more efficiently find deaminated 5mC bases [55,56,63,83,84]. The 280-residue linker joining the domains, which is likely disordered, could allow up to 50 bp separation between binding sites for the two domains [83]. NMR studies show that the MBD exchanges rapidly between 5mCG sites on DNA, which could enhance the efficiency of lesion search by full-length MBD4[83].

3.3 TDG-initiated BER

3.3.1 Specificity of TDG for deaminated 5mC

Human TDG is comprised of a catalytic domain (190 residues) flanked by two 110-residue regions that are largely disordered yet important for activity, protein interactions, and regulation by post-translational modifications such as acetylation, phosphorylation, and SUMO conjugation (Fig. 3A) [85–90]. TDG was discovered as an enzyme that excises T from G·T mispairsin vitro[91,92], as needed to repair 5mC deamination, and subsequent studies indicate that deaminated 5mC is one of its key biological targets [57,82]. For example, studies showing that depletion of MBD4 in mice gives an increase in CT transitions at CpG sites also suggested that another factor, likely TDG, also contributes to the repair of deaminated 5mC[62,66]. TDG is in fact the main G·T repair activity in cell-free extracts [93].More recently, loss of TDG was found to cause an increase in CT mutations at CpG sites in tumor cells from a patient exhibiting a deficiency in MMR[94].

Fig. 3.

Fig. 3

Structures and molecular dynamics (MD) studies of human TDG. (A) Schematic primary structure of TDG showing the catalytic (glycosylase) domain and the two flanking regions that are disordered yet functionally important. Shown are sites of post-translational modifications, the SUMO-interacting motif (SIM), and PIP degron that mediates interaction with PCNA and subsequent ubiquitination and degradation. (B) Structure of TDG (catalytic domain) bound to DNA with U of a G·U mispair flipped into active site (2.97 Å resolution, PDBID: 3UFJ). The DNA is shown as gray lines with surface representation; the G·U mispair is shown in cyan and the flanking C·G pair (i.e., CpG context) is yellow. Contacts with the flipped U and mismatched G are shown. (C) Structure of SUMO-1 modified TDG (residues 117–331, PDBID: 1WYW), with DNA modeled in (by aligning DNA-bound TDG; PDBID 2RBA). The SUMO-1 domain is covalently tethered to K330 of TDG and it forms many non-covalent interactions with the SIM of TDG (left surface as shown). SUMO-binding stabilizes an otherwise disordered helix of TDG (magenta), which likely weakens TDG binding to AP-DNA. (D) To illustrate a potential steric clash involving the methyl groups of a flipped thymine and Ala145, uracil from the structure in panel B was replaced with thymine (interactions shown are from the structure in panel B). (E) MD simulations of TDG bound to a G·T mispair suggest that the enzyme can transiently flip thymine into a position that lies just shy of the fully-flipped conformation which is likely needed for base excision. The last two panels are adapted from Ref [60].

Unlike the glycosylase domain of MBD4, TDG has stringent specificity for removing T bases that have a 3′ guanine (5′-TG-3′) [57,82], due likely to contacts it makes with the 3′-G [95,96]. This context specificity, unique for a glycosylase, provides full activity for G·T mispairs that arise by 5mC deamination. By contrast, activity will be weak for most G·T mispairs arising from replication errors, which is important because replication errors require action on the misincorporated nucleotide (G or T) via MMR processing of the nascent strand. Suppression of TDG activity during DNA replication is also accomplished by its degradation in S phase, as discussed below[97].

TDG activity is much greater for removing T from G·T relative to A·T pairs, as required for minimizing aberrant and mutagenic action on A·T pairs. Crystal structures suggest this specificity may derive in part from selective interactions with N1H and N2H2 of the mismatched guanine (Figs. 2E,3B)[60]. Remarkably, MBD4 and TDG both use backbone oxygen atoms to contact the same two nitrogen atoms of the mismatched guanine. However, the weaker and more dynamic base-pairing properties of G·T mispairs relative to A·T pairs could also contribute to mismatch specificity, as noted above for MBD4. Additional studies are needed to reveal how mismatch glycosylases act selectively on G·T mispairs.

3.3.2 Regulation of TDG by post-translational modifications

The catalytic turnover of TDG is severely impeded by tight binding to its AP-DNA product [98–102], a problem that could potentially be circumvented by post-translational modification [87]. TDG is modified by small ubiquitin-like modifier (SUMO) proteins at a single Lys residue (K330, human) [87], which weakens its binding to AP-DNA and depletes its G·T glycosylase activity[87,103,104]. It was proposed that selective modification ofproduct-bound TDG is required for efficient product release and subsequent substrate processing [87,105]. This model gained much attention [106,107], given that TDG is the only enzyme for which catalytic turnover is proposed to be regulated by sumoylation, and one of a few enzymes [108] whose activity is altered by SUMO. However, it was unknown whether SUMO conjugation is fast enough to hasten product release or specific forproduct-bound TDG. Recent studies show that modification by E2~SUMO is not selective for DNA-bound TDG, but it is relatively efficient such that it could potentially stimulate product release [109].

Sumoylation of TDG could potentially serve purposes other than (or in addition to) regulating product release. TDG has a SUMO interacting motif (SIM), located adjacent to theconjugation site, that binds noncovalently to SUMO proteins [104,105]. Many proteins that interact with TDG are themselves sumoylated and/or contain a SIM, including p53 and p300 [110–113]. Sumoylation of TDG could potentially stabilize its binding to proteins that have a SIM. Alternatively, sumoylation of TDG could hinder its binding to other sumoylated proteins. This is because the SUMO conjugation site of TDG (K330) flanks its own SIM, thus an intramolecular SUMO domain can occupy the SIM of TDG, as indicated by crystal structures (Fig. 3)[103,104]. This could explain findings that sumoylation of TDG suppresses its binding to free SUMO proteins [114] and hinders its association with (and acetylation by) p300 [113].

TDG is degraded in S phase by the ubiquitin-proteasome pathway [97], in a mechanism that requires PCNA and the E3 ubiquitin ligase CRL4Cdt2 [115,116]. Notably, TDG is also destroyed through the same process in response to DNA damage [115]. If not degraded, TDG slows S phase progression and cell proliferation[97,117]. The reason for S-phase degradation of TDG is not yet clear. One possibility is that TDG may generate and bind tightly to AP sites, which could block the replication fork and/or suppress efficient downstream repair, causing DNA strand breaks [97,115,117,118]. TDG might also initiate inappropriate (mutagenic) repair of G·T mismatches that arise by misincorporation of G during replication, which should be handled by MMR[97]. Alternatively, TDG might erroneously remove 5fC or 5caC during DNA replication, when heterochromatin is less condensed. In addition, it is possible that degradation of TDG is needed to regulate levels of DNA methylation during early stages of development, when most cells are undergoing DNA replication and division [115,116].

3.3.3 TDG repair is curtailed by residues that minimize action on undamaged DNA

While numerous findings indicate that G·T mispairs are a key biological target for TDG, its activity is weaker for these lesions relative to nearly all other substratesin vitro[57,98,119–122]. Many studies indicate that access to the TDG active site is limited for thymine and other bases with a bulky C-5 substituent, due likely to steric hindrance. A structure of TDG bound to a G·U mispair suggested a steric clash between the methyl groups of a flipped thymine base and Ala145 (Fig. 3D) [60]. Removing the methyl group of Ala145 (via A145G) yielded a 13-foldincrease in G·T activity, with no impact on G·U, as expected[60]. Consistent with these experimental findings, molecular dynamics (MD) simulations suggest that wild-type TDG stabilizes thymine in a partially flipped conformation that is not catalytically competent (Fig. 3E), whereas A145G-TDG exhibits complete thymine flipping [60].Unexpectedly, a second active-site residue also curtails repair by TDG; G·T activity is 13-foldhigher for H151A-TDG relative to native enzyme[60]. Why are two residues that curtail repair activity conserved in TDG enzymes (vertebrates)? One possibility is that they limit aberrant action on A·T pairs, which can be mutagenic and cytotoxic. Indeed, A·T activity is 38- and 34-fold higher for A145G- and H151A-TDG, respectively, compared to wild-type enzyme. To our knowledge, TDG is the only glycosylase for which repair activity is sharply curtailed by conserved active-site residues. It seems likely that this reflects a balance between the conflicting needs for efficient repair and minimal erroneous action on undamaged DNA. The suboptimal G·T repair activity of TDG could potentially account, in part, for the prevalence of CT mutations at CpG sites, which are among the most frequent mutations in cancer and genetic disease [61,123–126].

4. Role of base excision repair in active DNA demethylation

4.1 Overview

Many studies over the past two decades have suggested a potential role for MBD4 and TDG in active DNA demethylation, and the details have become increasingly clear in the last few years. Early reports suggested that both enzymes could directly remove 5mC from DNA [127,128], but subsequent studies demonstrate that their 5mC glycosylase activity is exceedingly weak and not biologically relevant[93,129,130]. Notably, 5mC glycosylase activity is found in plants [131]. More recent studies indicate that TDG and MBD4 act on derivatives of 5mC resulting from active deamination and/or oxidation (Fig. 1) [2]. The demethylation pathway involving TDG has been established biochemically and biologically, whereas the same cannot be said conclusively for MBD4.

4.2 A role for MBD4 in active DNA demethylation?

Raiet al reported in 2008 that MBD4 mediates active DNA demethylation in zebrafish embryos, by initiating base excision repair of G·T mispairs that are generated by activation-induced deaminase (AID), and that Gadd45 facilitates a functional interaction between MBD4 and AID [132]. However, a recent report concludes that key findings of Raiet al are not reproducible and that there is no evidence for AID-MBD4 mediated DNA demethylation in zebrafish embryos [133]. Another study reported a role for MBD4 in hormone-induced DNA demethylation in in mice [134], but this paper was subsequently retracted [135]. More recently, Sabaget al proposed that a TET1-AID-MBD4-Gadd45 pathway mediates DNA demethylation during reprogramming of somatic cells to generate induced pluripotent stem (iPS) cells [136]. Thus, biological studies have not conclusively demonstrated a role for MBD4 in active DNA demethylation.

In addition, biochemical studies raise questions about the role of MBD4 in DNA demethylation. Because MBD4 cannot excise 5mC, 5hmC, 5fC or 5caC[55,56,74,93], an MBD4-mediated pathway would necessitate that a deaminase of the AID-Apolipoprotein B RNA-editing catalytic component (APOBEC) family convert 5mC to T, or 5hmC to 5hmU, to generate a substrate for MBD4 (G·T or G·5hmU). However, the plausibility of a pathway involving 5hmC deamination is questioned by findings that deamination of 5hmC by AID or APOBEC enzymes is not detectablein vitro or in cells [137,138]. While deamination of 5hmC occursin vitro, it is ~10-fold slower than deamination of C. Additionally, the preference of AID-APOBEC enzymes for single stranded DNA and for particular DNA sequences, together with findings that deaminase deficiency causes no significant developmental defects (with the caveat of possible redundancy), suggest a limited role for these enzymes in 5mC deamination [1,2]. Even if deamination of 5mC or 5hmC is efficientin vivo, findings that MBD4-deficient mice are viable and exhibit no obvious developmental or DNA methylation defects also argues against a substantial role for a deaminase-MBD4-BER pathway for DNA demethylation. Thus, compared to the TET-TDG pathway, discussed below, the (TET)-deaminase-MBD4 pathway for DNA demethylation is not well established byin vitro orin vivo studies reported to date.

4.3 TDG in active DNA demethylation and transcriptional regulation

TDG plays complex roles in DNA demethylation and transcriptional regulation, through both catalytic and non-catalytic (structural) mechanisms. Recent genetic and biochemical studies have converged to provide strong evidence for a prominent role of TDG in DNA demethylation and epigenetic regulation. First, mice with targeted inactivation of TDG in the germ line revealed embryonic lethality and developmental abnormalities associated with hypermethylation of promoter CpG islands[93,130].In addition, demethylation of tissue specific, developmentally regulated enhancers was impaired in the absence of TDG[93]. More importantly, knock-in of a catalytically inactive TDG variant also manifested embryonic lethality in mice, and reproduced the enhancer demethylation defect, thus providing compelling genetic evidence for an active and enzymatically-driven process for DNA demethylation [93].

In parallel, biochemical studies have begun to clarify the mechanistic role of TDG in DNA demethylation. TDG has robust glycosylase activity on 5fC and 5caC opposite G, and the levels of these epigenetic bases increase upon TDG knock-down, thus implying a necessary role for TDG acting downstream of TET enzymes in a pathway for DNA demethylation (Fig. 1) [29,31,96,139,140].Whereas the TET-TDG-BER appears to be the main demethylation pathway, TDG may play a role in alternative pathways as well. Although the involvement of AID/APOBEC deaminasesin DNA demethylation is controversial, TDG could potentially act on G·T mismatches originated by enzymatic deamination of 5mC. In fact, a physical interaction was detected between AID and TDG [93]. Although it is unlikely that AID-APOBEC deaminases mediate deamination of 5hmC to 5hmU due to the increased hindrance of 5hmC in comparison to 5mC and C [137], TDG can excise hmU [93,129,141].

In addition to its catalytic role, TDG may affect DNA demethylation and transcriptional activation in other ways, such as a structural adaptor/scaffold or a co-activator. In fact, it is known that active transcription contributes to maintaining CpG islands in their unmethylated state [6,142], and it is possible that TDG is involved in promoting an open chromatin environment via protein-protein interactions. TDG interacts with DNMT3a and DNMT3b; as a result of these interactions, the two DNMTs stimulate TDG glycosylase activity whereas TDG inhibits DNMT3a [143,144].TDG also interacts with and is acetylated by the histone acetyl-transferases p300 and CREB-binding protein (CBP) [85], and is necessary for recruitment of p300 and MLL to active promoters [93,130]. Due to this transcriptional co-activator role, TDG modulates the activity of several nuclear hormone receptors, including estrogen receptor alpha (ERα, retinoic acid and retinoic X receptors, and the transcriptional activators p300, CBP and p160 [85,93,145–147]. Thus, the absence of TDG is associated with the loss of activating histone marks (H3K9ac, H3K14ac, H3K4me2) and an increase of repressive histone marks (H3K27me3 and H3K9me3) [93,130]. In the future, it will be important to define more precisely the coordination of the catalytic and non-catalytic functions of TDG in transcriptional control.

5. Perspectives and Future Challenges

The last two decades have seen an increased comprehension of how BER maintains the genetic and epigenetic stability of CpG sites. Indeed, studies over the past five years have resulted in the characterization of active DNA demethylation, the identification of novel substrates of MBD4 and TDG, and the determination of DNA-bound structures or these enzymes. Nevertheless, many important challenges remain for the years ahead.

The respective roles of MBD4 and TDG in genomic and epigenomic integrity need to be established: for the shared substrates (G·T, G·U, G·hmU), do the two enzymes exhibit redundant activities or do they protect distinct areas of the genome at different stages of life(development, adult) and/or indifferent tissues?

At the moment, the available data appear to indicate a role for TDG, but not MBD4, in development and active DNA demethylation. Is it possible, however, that MBD4 has limited but defined roles in developmental control and cytosine demethylation? The same can be said for the mechanisms of DNA demethylation: most of the evidence indicates that the TET-TDG pathway is prevalent. However, it may be premature to rule out any role for deaminases in the process.

It is important to fully characterize the epigenetic roles of 5fC and 5caC, in addition to serving intermediates for cytosine demethylation, as is the case for 5hmC. This and other knowledge is likely to come from the improvement and widespread application of techniques for single-base resolution mapping of these bases, among other methods[34,148,149].

It is certainly an exciting time to study the connections between the genetic and epigenetic integrity of CpG sites. The next decade will allow an exploration of these open questions, coupled with a better understanding of BER functions in cancer and genetic diseases and hopefully with potential therapeutic applications of this knowledge.

Acknowledgments

We would like to thank past and present members of our respective groups for feedback and open discussions over the years; and R. Sonlinand R. Brooks for secretarial assistance. The authors apologize to colleagues for having failed to cite their work due to space constraints. Work in the authors’ laboratories is supported by National Cancer Institute grant CA078412 (to AB), National Institute of General Medical Sciences grant GM072711 (to ACD), National Cancer Institute grant CA06927 to Fox Chase Cancer Center, a grant with the Pennsylvania Department of Health, and an appropriation from the Commonwealth of Pennsylvania.

Footnotes

Conflict of interest statement

The authors declare no conflict of interest

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