Monoubiquitinated histone H2B plays multiple roles in transcription activation. H2B is deubiquitinated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator, which contains a four-protein subcomplex known as the deubiquitinating (DUB) module. The crystal structure of the Ubp8/Sgf11/Sus1/Sgf73 DUB module bound to a ubiquitinated nucleosome reveals that the DUB module primarily contacts H2A/H2B, with an arginine cluster on the Sgf11 zinc finger domain docking on the conserved H2A/H2B acidic patch. The Ubp8 catalytic domain mediates additional contacts with H2B, as well as with the conjugated ubiquitin. We find that the DUB module deubiquitinates H2B both in the context of the nucleosome and in H2A/H2B dimers complexed with the histone chaperone, FACT, suggesting that SAGA could target H2B at multiple stages of nucleosome disassembly and reassembly during transcription.

Cytosine methylation at CpG dinucleotides produces m 5 CpG, an epigenetic modification that is important for transcriptional regulation and genomic stability in vertebrate cells. However, m 5 C deamination yields mutagenic G⅐T mispairs, which are implicated in genetic disease, cancer, and aging. Human thymine DNA glycosylase (hTDG) removes T from G⅐T mispairs, producing an abasic (or AP) site, and follow-on base excision repair proteins restore the G⅐C pair. hTDG is inactive against normal A⅐T pairs, and is most effective for G⅐T mispairs and other damage located in a CpG context. The molecular basis of these important catalytic properties has remained unknown. Here, we report a crystal structure of hTDG (catalytic domain, hTDG cat ) in complex with abasic DNA, at 2.8 Å resolution. Surprisingly, the enzyme crystallized in a 2:1 complex with DNA, one subunit bound at the abasic site, as anticipated, and the other at an undamaged (nonspecific) site. Isothermal titration calorimetry and electrophoretic mobility-shift experiments indicate that hTDG and hTDG cat can bind abasic DNA with 1:1 or 2:1 stoichiometry. Kinetics experiments show that the 1:1 complex is sufficient for full catalytic (base excision) activity, suggesting that the 2:1 complex, if adopted in vivo, might be important for some other activity of hTDG, perhaps binding interactions with other proteins. Our structure reveals interactions that promote the stringent specificity for guanine versus adenine as the pairing partner of the target base and interactions that likely confer CpG sequence specificity. We find striking differences between hTDG and its prokaryotic ortholog (MUG), despite the relatively high (32%) sequence identity.CpG site ͉ DNA repair ͉ G⅐T mismatch ͉ deamination ͉ 5-methylcytosine H uman thymine DNA glycosylase (hTDG) belongs to the uracil DNA glycosylase (UDG) superfamily of enzymes that share a common ␣/␤ fold and promote genomic integrity by removing mutagenic uracil bases from DNA (1, 2). Initiating the base excision repair pathway, these enzymes use a remarkable nucleotide-flipping mechanism to extrude damaged nucleobases from the DNA helix and cleave the base-sugar (N-glycosidic) bond, producing an abasic (or AP) site in the DNA (3). Together, hTDG and the Escherichia coli mismatch-specific uracil DNA glycosylase (eMUG) are the most thoroughly characterized members of the TDG/MUG family (4-6). These enzymes excise a variety of damaged bases (X), and typically exhibit a strong preference for lesions in G⅐X versus A⅐X pairs (7-12). Like its eukaryotic orthologs, hTDG (410 residues) contains a conserved catalytic core (residues 121-300) flanked by more divergent Nand C-terminal domains (6); the former enhances DNA binding and G⅐T repair activity to some extent (13,14), and the latter contains a site for SUMO conjugation (K330), a modification that decreases the DNA-binding affinity of hTDG (15,16).A recent structure of the hTDG catalytic domain (residues 117-332, conjugated to SUMO-1) reveals strong similarity to the structure of...

Thymine DNA glycosylase (TDG) excises thymine from G⅐T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG⅐X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG⅐FU, CpG⅐ClU, and CpG⅐BrU than for CpG⅐T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k max ) for G⅐X substrates depends significantly on the 5 base pair. For example, k max decreases by 6-, 11-, and 82-fold for TpG⅐ClU, GpG⅐ClU, and ApG⅐ClU, respectively, as compared with CpG⅐ClU. For the other G⅐X substrates, the 5-neighbor effects have a similar trend but vary in magnitude. The activity for G⅐FU, G⅐ClU, and G⅐BrU, with any 5-flanking pair, meets and in most cases significantly exceeds the CpG⅐T activity. Strikingly, human TDG activity is reduced 10 2.3 -10 4.3 -fold for A⅐X relative to G⅐X pairs and reduced further for A⅐X pairs with a 5 pair other than C⅐G. The effect of altering the 5 pair and/or the opposing base (G⅐X versus A⅐X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.The nucleobases in DNA are subject to continuous chemical modification, generating a broad range of mutagenic and cytotoxic lesions that can lead to cancer and other diseases (1, 2). To counteract this inevitable damage, the cellular machinery includes systems for DNA repair (3). Damage occurring to the nucleobases is the purview of base excision repair, a pathway that is initiated by a damage-specific DNA glycosylase. These enzymes find damaged or mismatched bases within the vast expanse of normal DNA and catalyze the cleavage of the basesugar (N-glycosidic) bond, producing an abasic or apurinic/ apyrimidinic (AP) 2 site in the DNA. The repair process is continued by follow-on base excision repair enzymes.Human thymine DNA glycosylase (hTDG) was discovered as an enzyme that removes thymine from G⅐T and uracil from G⅐U mispairs in DNA (4, 5). In vertebrates, G⅐T mispairs arise from replication errors, which are handled by the mismatch repair pathway or from the deamination of 5-methylcytosine to T (6, 7). Because cytosine methylation occurs at CpG dinucleotides (8, 9), G⅐T mispairs caused by 5-methylcytosine deamination are found at CpG sites. It has been shown that hTDG is most active for G⅐T mispairs with a 5Ј C⅐G pair, suggesting that a predominant biological role of the enzyme is to initiate the repair of CpG⅐T lesions (10, 11). DNA methylation at CpG plays a fundamental role in many cellular processes, including transcriptional regulation and the silencing of repetitive genetic elements (8, 9). Sugg...

Thymine DNA glycosylase (TDG) promotes genomic integrity by excising thymine from mutagenic G⅐T mismatches arising by deamination of 5-methylcytosine, and follow-on base excision repair enzymes restore a G⅐C pair. TDG cleaves the N-glycosylic bond of dT and some other nucleotides, including 5-substituted 2-deoxyuridine analogs, once they have been flipped from the helix into its active site. We examined the role of two strictly conserved residues; Asn 140 , implicated in the chemical step, and Arg 275 , implicated in nucleotide flipping. The N140A variant binds substrate DNA with the same tight affinity as wild-type TDG, but it has no detectable base excision activity for a G⅐T substrate, and its excision rate is vastly diminished (by ϳ10 4.4 -fold) for G⅐U, G⅐FU, and G⅐BrU substrates. Thus, Asn 140 does not contribute substantially to substrate binding but is essential for the chemical step, where it stabilizes the transition state by ϳ6 kcal/mol (compared with 11.6 kcal/ mol stabilization provided by TDG overall). Our recent crystal structure revealed that Arg 275 penetrates the DNA minor groove, filling the void created by nucleotide flipping. We found that the R275A and R275L substitutions weaken substrate binding and substantially decrease the base excision rate for G⅐T and G⅐BrU substrates. Our results indicate that Arg 275 promotes and/or stabilizes nucleotide flipping, a role that is most important for target nucleotides that are relatively large (dT and bromodeoxyuridine) and/or have a stable N-glycosylic bond (dT). Arg 275 does not contribute substantially to the binding of TDG to abasic DNA product, and it cannot account for the slow product release exhibited by TDG.The chemically reactive bases in DNA are continuously modified by oxidation, alkylation, and deamination, creating mutagenic and cytotoxic lesions that are implicated in aging and diseases including cancer (1, 2). Such damage is handled predominantly by the base excision repair pathway, initiated by one of many damage-specific DNA glycosylases (3). These enzymes use a nucleotide flipping mechanism to find damaged bases within the vast excess of normal DNA and cleave the base-sugar (N-glycosylic) bond to release the base, and follow-on base excision repair proteins complete the repair process.Thymine DNA glycosylase (TDG) 2 removes thymine from mutagenic G⅐T mispairs, one of the few glycosylases that removes a normal base from DNA. Consistent with the need to avoid acting upon undamaged DNA, TDG activity is 18,000-fold greater for G⅐T mispairs relative to A⅐T pairs (4). TDG is also specific for a particular DNA sequence, exhibiting the highest activity for G⅐T mispairs (and other lesions) with a