Role of MED1 (MBD4) Gene in DNA repair and human cancer

The human protein MED1, also known as MBD4, was isolated in a yeast two-hybrid screening as an interactor of the mismatch repair protein MLH1. MED1 contains an N-terminal 5-methylcytosine binding domain (MBD), which allows binding to methylated DNA, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. This suggests that DNA methylation may play a role in human DNA repair. MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, 5-fluorouracil and, weakly, 3,N(4)-ethenocytosine paired with guanine. The glycosylase activity of MED1 prefers substrates in which the G:T mismatch is present in the context of methylated or unmethylated CpG sites. Since G:T mismatches can originate via spontaneous deamination of 5-methylcytosine to thymine, MED1 appears to act as a caretaker of genomic fidelity at CpG sites. Mutagenesis caused by these deamination events is a frequent mechanism of genetic instability in cancer; thus, based on the biochemical activity of its gene product, MED1 is a candidate tumor suppressor gene. Indeed, frameshift mutations of the MED1 gene have been reported in human colorectal, gastric, endometrial, and pancreatic cancer. In the future, efforts should be directed toward investigations of the functional role of the MED1 gene in the pathogenesis, prevention, and treatment of human cancer.     

Investigation of the substrate spectrum of the human mismatch-specific DNA N-glycosylase MED1 (MBD4): fundamental role of the catalytic domain

The human DNA repair protein MED1 (also known as MBD4) was isolated as an interactor of the mismatch repair protein MLH1 in a yeast two-hybrid screening. MED1 has a tripartite structure with an N-terminal 5-methylcytosine binding domain (MBD), a central region, and a C-terminal catalytic domain with homology to bacterial DNA damage-specific glycosylases/lyases. Indeed, MED1 acts as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil paired with guanine. The glycosylase activity of MED1 preferentially targets G:T mismatches in the context of CpG sites; this indicates that MED1 is involved in the repair of deaminated 5-methylcytosine. Interestingly, frameshift mutations of the MED1 gene have been reported in human colorectal, endometrial, and pancreatic cancers. For its putative role in maintaining genomic fidelity at CpG sites, it is important to characterize the biochemical properties and the substrate spectrum of MED1. Here we show that MED1 works under a wide range of temperature and pH, and has a limited optimum range of ionic strength. MED1 has a weak glycosylase activity on the mutagenic adduct 3,N(4)-ethenocytosine, a metabolite of vinyl chloride and ethyl carbamate. The differences in glycosylase activity on G:U and G:T substrates are not related to differences in substrate binding and likely result from intrinsic differences in the chemical step. Finally, the isolated catalytic domain of MED1 retains the preference for G:T and G:U substrates in the context of methylated or unmethylated CpG sites. This suggests that the catalytic domain is fundamental, and the 5-methylcytosine binding domain dispensable, in determining the substrate spectrum of MED1.     

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.     

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD–DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16^INK4a genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.     

The S. pombe Translation Initiation Factor eIF4G Is Sumoylated and Associates with the SUMO Protease Ulp2

SUMO is a small post-translational modifier, that is attached to lysine residues in target proteins. It acts by altering protein-protein interactions, protein localisation and protein activity. SUMO chains can also act as substrates for ubiquitination, resulting in proteasome-mediated degradation of the target protein. SUMO is removed from target proteins by one of a number of specific proteases. The processes of sumoylation and desumoylation have well documented roles in DNA metabolism and in the maintenance of chromatin structure. To further analyse the role of this modification, we have purified protein complexes containing the S. pombe SUMO protease, Ulp2. These complexes contain proteins required for ribosome biogenesis, RNA stability and protein synthesis. Here we have focussed on two translation initiation factors that we identified as co-purifying with Ulp2, eIF4G and eIF3h. We demonstrate that eIF4G, but not eIF3h, is sumoylated. This modification is increased under conditions that produce cytoplasmic stress granules. Consistent with this we observe partial co-localisation of eIF4G and SUMO in stressed cells. Using HeLa cells, we demonstrate that human eIF4GI is also sumoylated; in vitro studies indicate that human eIF4GI is modified on K1368 and K1588, that are located in the C-terminal eIF4A- and Mnk-binding sites respectively.     

A SUMO-interacting motif activates budding yeast ubiquitin ligase Rad18 towards SUMO-modified PCNA

SUMO-targeted ubiquitin ligases (STUbLs) recognize sumoylated proteins as substrates for ubiquitylation and have been implicated in several aspects of DNA repair and the damage response. However, few physiological STUbL substrates have been identified, and the relative importance of SUMO binding versus direct interactions with the substrate remains a matter of debate. We now present evidence that the ubiquitin ligase Rad18 from Saccharomyces cerevisiae, which monoubiquitylates the sliding clamp protein proliferating cell nuclear antigen (PCNA) in response to DNA damage, exhibits the hallmarks of a STUbL. Although not completely dependent on sumoylation, Rad18’s activity towards PCNA is strongly enhanced by the presence of SUMO on the clamp. The stimulation is brought about by a SUMO-interacting motif in Rad18, which also mediates sumoylation of Rad18 itself. Our results imply that sumoylated PCNA is the physiological ubiquitylation target of budding yeast Rad18 and suggest a new mechanism by which the transition from S phase-associated sumoylation to damage-induced ubiquitylation of PCNA is accomplished.     

Pellino-1, an adaptor protein of interleukin-1 receptor/toll-like receptor signaling,is sumoylated by Ubc9

Covalent modifications of the Pellino-1 protein are essential for transmitting innate immune response signals downstream, as the phosphorylation and polyubiquitination of Pellino-1 mediated by the IRAK proteins appear to have roles in regulating Pellino-1 function. In this study, we demonstrate that the Pellino-1 protein is post-translationally modified by small-ubiquitin-related modifier-1 (SUMO-1). Sumoylation assays with Pellino-1 and SUMO-1 expression plasmids reveal that the Pellino-1 protein is sumoylated in vitro and in vivo. Treatment of SUMO-1 specific protease 1 (SENP1) inhibited the sumoylation of the Pellino-1 protein and a GST pull-down assay as well as a yeast two hybrid assay showed that Pellino-1 binds to the SUMO-conjugating enzyme, Ubc9. Furthermore, we identified the five lysine residues of the Pellino-1 protein where SUMO-1 covalently attaches. Some of the sumoylated sites overlap with previously identified ubiquitination sites, suggesting competition between sumoylation and ubiquitination, as well as suggesting that the sumoylated Pellino-1 protein may have a cellular function distinct from previously identified functions.     

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.     

SUMO-targeted ubiquitin ligases in genome stability

We identify the SUMO-Targeted Ubiquitin Ligase (STUbL) family of proteins and propose that STUbLs selectively ubiquitinate sumoylated proteins and proteins that contain SUMO-like domains (SLDs). STUbL recruitment to sumoylated/SLD proteins is mediated by tandem SUMO interaction motifs (SIMs) within the STUbLs N-terminus. STUbL-mediated ubiquitination maintains sumoylation pathway homeostasis by promoting target protein desumoylation and/or degradation. Thus, STUbLs establish a novel mode of communication between the sumoylation and ubiquitination pathways. STUbLs are evolutionarily conserved and include: Schizosaccharomyces pombe Slx8-Rfp (founding member), Homo sapiens RNF4, Dictyostelium discoideum MIP1 and Saccharomyces cerevisiae Slx5-Slx8. Cells lacking Slx8-Rfp accumulate sumoylated proteins, display genomic instability, and are hypersensitive to genotoxic stress. These phenotypes are suppressed by deletion of the major SUMO ligase Pli1, demonstrating the specificity of STUbLs as regulators of sumoylated proteins. Notably, human RNF4 expression restores SUMO pathway homeostasis in fission yeast lacking Slx8-Rfp, underscoring the evolutionary functional conservation of STUbLs. The DNA repair factor Rad60 and its human homolog NIP45, which contain SLDs, are candidate STUbL targets. Consistently, Rad60 and Slx8-Rfp mutants have similar DNA repair defects.     

Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair

While methylcytosines serve as the fifth base encoding epigenetic information, they are also a dangerous endogenous mutagen due to their intrinsic instability. Methylcytosine undergoes spontaneous deamination, at a rate much higher than cytosine, to generate thymine. In mammals, two repair enzymes, thymine DNA glycosylase (TDG) and methyl-CpG binding domain 4 (MBD4), have evolved to counteract the mutagenic effect of methylcytosines. Both recognize G/T mismatches arising from methylcytosine deamination and initiate base-excision repair that corrects them to G/C pairs. However, the mechanism by which the methylation status of the repaired cytosines is restored has remained unknown. We show here that the DNA methyltransferase Dnmt3a interacts with TDG. Both the PWWP domain and the catalytic domain of Dnmt3a are able to mediate the interaction with TDG at its N-terminus. The interaction affects the enzymatic activity of both proteins: Dnmt3a positively regulates the glycosylase activity of TDG, while TDG inhibits the methylation activity of Dnmt3a in vitro. These data suggest a mechanistic link between DNA repair and remethylation at sites affected by methylcytosine deamination.     

Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil-containing DNA

Active DNA demethylation in mammals occurs via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation family of proteins (TETs). 5hmC residues in DNA can be further oxidized by TETs to 5-carboxylcytosines and/or deaminated by the Activation Induced Deaminase/Apolipoprotein B mRNA-editing enzyme complex family proteins to 5-hydromethyluracil (5hmU). Excision and replacement of these intermediates is initiated by DNA glycosylases such as thymine-DNA glycosylase (TDG), methyl-binding domain protein 4 (MBD4) and single-strand specific monofunctional uracil-DNA glycosylase 1 in the base excision repair pathway. Here, we report detailed biochemical and structural characterization of human MBD4 which contains mismatch-specific TDG activity. Full-length as well as catalytic domain (residues 426–580) of human MBD4 (MBD4^cat) can remove 5hmU when opposite to G with good efficiency. Here, we also report six crystal structures of human MBD4^cat: an unliganded form and five binary complexes with duplex DNA containing a T•G, 5hmU•G or AP•G (apurinic/apyrimidinic) mismatch at the target base pair. These structures reveal that MBD4^cat uses a base flipping mechanism to specifically recognize thymine and 5hmU. The recognition mechanism of flipped-out 5hmU bases in MBD4^cat active site supports the potential role of MBD4, together with TDG, in maintenance of genome stability and active DNA demethylation in mammals.     

Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase

The human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine. MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.