In addition to its well-documented effects on gene silencing, cytosine methylation is a prominent cause of mutations. In humans, the mutation rate from 5-methylcytosine (m5C) to thymine (T) is 10-50-fold higher than other transitions and the methylated sequence CpG is consequently under-represented. Over one-third of germline point mutations associated with human genetic disease and many somatic mutations leading to cancer involve loss of CpG. The primary cause of mutability appears to be hydrolytic deamination. Cytosine deamination produces mismatched uracil (U), which can be removed by uracil glycosylase, whereas m5C deamination generates a G x T mispair that cannot be processed by this enzyme. Correction of m5CpG x TpG mismatches may instead be initiated by the thymine DNA glycosylase, TDG. Here we show that MBD4, an unrelated mammalian protein that contains a methyl-CpG binding domain, can also efficiently remove thymine or uracil from a mismatches CpG site in vitro. Furthermore, the methyl-CpG binding domain of MBD4 binds preferentially to m5CpG x TpG mismatches-the primary product of deamination at methyl-CpG. The combined specificities of binding and catalysis indicate that this enzyme may function to minimize mutation at methyl-CpG.

U.Hardeland and R.Steinacher contributed equally to this work DNA glycosylases initiate base excision repair (BER) through the generation of potentially harmful abasic sites (AP sites) in DNA. Human thymine-DNA glycosylase (TDG) is a mismatch-speci®c uracil/thymine-DNA glycosylase with an implicated function in the restoration of G´C base pairs at sites of cytosine or 5-methylcytosine deamination. The rate-limiting step in the action of TDG in vitro is its dissociation from the product AP site, suggesting the existence of a speci®c enzyme release mechanism in vivo. We show here that TDG interacts with and is covalently modi®ed by the ubiquitin-like proteins SUMO-1 and SUMO-2/3. SUMO conjugation dramatically reduces the DNA substrate and AP site binding af®nity of TDG, and this is associated with a signi®cant increase in enzymatic turnover in reactions with a G´U substrate and the loss of G´T processing activity. Sumoylation also potentiates the stimulatory effect of APE1 on TDG. These observations implicate a function of sumoylation in the controlled dissociation of TDG from the AP site and open up novel perspectives for the understanding of the molecular mechanisms coordinating the early steps of BER.

The ubiquitin-related protein SUMO-1 is covalently attached to proteins by SUMO-1 ligases. We have performed a proteome-wide analysis of sumoylated substrate proteins in yeast. Employing the powerful affinity purification of Protein A-Smt3 (Smt3 is the yeast homologue of SUMO-1) from yeast lysates in combination with tandem liquid chromatography mass spectrometry, we have isolated potential Smt3-carrying substrate proteins involved in DNA replication and repair, chromatin remodeling, transcription activation, Pol-I, Pol-II, and Pol-III transcription, 5 pre-mRNA capping, 3 pre-mRNA processing, proteasome function, and tubulin folding. Employing tandem affinity purifications or a rapid biochemical assay referred to as "SUMO fingerprint," we showed that several subunits of RNA polymerases I, II, and III, members of the transcription repression and chromatin remodeling machineries previously not known to be sumoylated, are modified by SUMO-1. Thus, the identification of a broad range of SUMO-1 substrate proteins is expected to lead to further insight into the regulatory aspects of sumoylation.The ubiquitin-like protein SUMO-1 (Smt3 in yeast) is a 100-residue protein that is conjugated to substrate proteins by sequential thioester transfer reactions via specific E1 1 activating enzymes (Uba2/Aos1) and E2 (Ubc9) conjugating enzyme (1, 2). SUMO is conjugated to specific lysine residues on substrate proteins typically exhibiting consensus sites hKxE, where h is a hydrophobic amino acid (3, 4). Unlike ubiquitylation, sumoylation of target proteins does not lead to proteasomal degradation but can affect diverse functions of the protein, such as subcellular localization, protein/DNA interaction, or enzymatic activity (5, 6).Smt3 is highly conserved and essential in yeast. In addition, the conjugating and deconjugating machinery are conserved from yeast to humans and perform essential functions in yeast. The only yeast proteins known to be modified by Smt3 are the nonessential septins involved in cytokinesis and the essential Pol30, Top2, and Pds5 (7-9). A growing number of proteins that are modified by SUMO-1 in mammalian cells have been reported (6). Recent proteomic approaches in mammalian cells have identified new proteins that are subject to SUMO-1 and SUMO-2 modification (10 -12). The confirmation of sumoylation of target proteins in these approaches was limited by the need for protein specific antibodies.Previously, we have shown that the deconjugating enzyme Ulp1 is tethered to the nuclear pore channel via nuclear import receptors (13). The biological significance of such a tethering mechanism is poorly understood because of the lack of knowledge about its substrate proteins. Yeast has served as a powerful, rapid, genetic and in vivo biochemical model system to gain mechanistic insights into protein function in eukaryotes. Hence, we applied a proteomic approach in yeast to unravel the SUMO proteome. We have enriched sumoylated proteins from yeast cell lysates using the high affinity tag of Protein A (ProtA). In this...

Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine-or cytosine deamination in DNA by its ability to release thymine or uracil from G⅐T and G⅐U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G⅐U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.DNA of all organisms is susceptible to modification and damage through the action of a variety of exogenous and endogenous reagents. A prominent form of spontaneous damage arises through hydrolytic deamination of bases carrying exocyclic amino groups such as cytosine and 5-methylcytosine. Deamination of cytosine in double-stranded DNA (dsDNA) 1 generates a uracil⅐guanine mispair and, similarly, deamination of 5-methycytosine generates a thymine⅐guanine mispair. In vitro, both events occur at appreciable rates, with 5-methylcytosine deamination being slightly faster than that of cytosine (1) and, in vivo, both deamination products are mutagenic and will produce C3 T transitions upon DNA replication, if left unrepaired. Whereas accurate repair of G⅐U mispairs is mediated by enzymes that specifically recognize and process uracil in DNA, e.g. uracil DNA glycosylase (UDG) (2), correction of G⅐T mispairs to G⅐C base pairs requires a repair function that is able to discriminate between a mutagenic thymine in a G⅐T mismatch and a normal thymine base-paired with adenine.A G⅐T mismatch-specific thymine glycosylase activity was discovered in HeLa cell extracts (3). It was purified to apparent homogeneity (4), and the encoding cDNA was cloned (5). The biochemical properties of this thymine DNA glycosylase (TDG) are compatible with a function of the enzyme in cellular defense against mutagenesis by cytosine and 5-methylcytosine deamination. It is capable of recognizing G⅐T and G⅐U mismatches in DNA and initiating their restoration to G⅐C base pairs through a base excision repair process involving DNA polymerase ␤ (6 -8). Two bacterial open reading frames with significant homology to the central part of hum...

Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5'-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species.

Inflammatory responses represent a hallmark of numerous pathologies including sepsis, bacterial infection, insulin resistance, and malign obesity. Here we describe an unexpected coactivator function for the nuclear receptor interacting protein 140 (RIP140) for nuclear factor B (NFB), a master transcriptional regulator of inflammation in multiple tissues. Previous work has shown that IntroductionMetabolic diseases, such as insulin resistance, obesity, and atherosclerosis, have recently been recognized as low-grade, subacute inflammatory conditions, contributing to the development of type II diabetes and cardiovascular failure. 1 Similar to acute inflammation, all of these conditions are characterized by elevated levels of proinflammatory cytokines such as interleukin-1␤ (IL-1␤) and IL-6, and tumor necrosis factor ␣ (TNF␣). 2,3 Toward this end, levels of IL-6, IL-1␤, and TNF␣ are elevated in obese patients and mouse models of insulin resistance and obesity. [2][3][4] In this respect, ablation of the TNF␣ gene or of its receptor renders mice resistant to the development of insulin resistance and associated metabolic disorders. 5,6 A common polymorphism has been identified in the IL-6 receptor gene that is associated with energy intake and obesity in humans, 7 underlining the critical impact of cytokine signaling for metabolic diseases.The inflammatory response emerging in the presence of insulin resistance and obesity seems to reside predominantly in adipose tissue. 1,8 Indeed, transgenic overexpression of monocyte chemotactic protein 1 (MCP1) in adipose tissue results in enhanced macrophage infiltration, inflammation, and insulin resistance. 9,10 On the other hand, impairment of macrophage migration into adipose tissue by genetic knockout of the MCP1 receptor chemotactic cytokine receptor 2 (CCR2) has been found to substantially improve tissue inflammation and insulin sensitivity. 10,11 Importantly, macrophages have recently been shown to accumulate under obese conditions in adipose tissue of both mice and humans. 12,13 Resident macrophages in adipose tissue, therefore, seem to be in large part responsible for the cytokine release of this tissue and the systemic inflammation associated with obesity. 12,14 These studies highlighted the importance of adipose tissue as a key site for the interaction of metabolic cells with effectors of the immune system, specifically macrophages, to control systemic energy homeostasis and to trigger metabolic dysfunction under pathophysiologic conditions. 8 Consequently, not only adipose tissue quantity but also quality, as exemplified by its macrophage content and inflammatory status, seems to represent a critical determinant for the onset of insulin resistance and other components of the metabolic syndrome. 8,15 In this respect, the cytokine release from macrophages in response to external signals is largely determined by transcriptional mechanisms. A number of transcriptional regulators have been identified as critical checkpoints for the proinflammatory response of macrophages, ...

We describe a novel regulatory mechanism for DNA polymerase beta (Polbeta), a protein involved in DNA base excision repair (BER). Polbeta colocalized in vivo and formed a complex with the transcriptional coactivator p300. p300 interacted with Polbeta through distinct domains and acetylated Polbeta in vitro. Polbeta acetylation was furthermore observed in vivo. Lysine 72 of Polbeta was identified as the main target for acetylation by p300. Interestingly, acetylated Polbeta showed a severely reduced ability to participate in a reconstituted BER assay. This was due to an impairment of the dRP-lyase activity of Polbeta. Acetylation of Polbeta thus acts as an intranuclear regulatory mechanism and implies that p300 plays a critical regulatory role in BER.

Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin–proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin–proteasome pathway constitutes the underlying regulatory system.