Repair of a uracil‐guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil‐DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two‐hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double‐stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84–183 in the N‐terminal half of the XRCC1 protein, whereas the C‐terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision‐repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell‐free extracts of an XRCC1‐deficient mutant cell line, in agreement with the results from the reconstituted system.

Thymine DNA glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily of DNA repair enzymes. Owing to its ability to excise thymine when mispaired with guanine, it was proposed to act against the mutability of 5-methylcytosine (5-mC) deamination in mammalian DNA. However, TDG was also found to interact with transcription factors, histone acetyltransferases and de novo DNA methyltransferases, and it has been associated with DNA demethylation in gene promoters following activation of transcription, altogether implicating an engagement in gene regulation rather than DNA repair. Here we use a mouse genetic approach to determine the biological function of this multifaceted DNA repair enzyme. We find that, unlike other DNA glycosylases, TDG is essential for embryonic development, and that this phenotype is associated with epigenetic aberrations affecting the expression of developmental genes. Fibroblasts derived from Tdg null embryos (mouse embryonic fibroblasts, MEFs) show impaired gene regulation, coincident with imbalanced histone modification and CpG methylation at promoters of affected genes. TDG associates with the promoters of such genes both in fibroblasts and in embryonic stem cells (ESCs), but epigenetic aberrations only appear upon cell lineage commitment. We show that TDG contributes to the maintenance of active and bivalent chromatin throughout cell differentiation, facilitating a proper assembly of chromatin-modifying complexes and initiating base excision repair to counter aberrant de novo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.

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 base excision repair machinery protects DNA in cells from the damaging effects of oxidation, alkylation, and deamination; it is specialized to fix single-base damage in the form of small chemical modifications. Base modifications can be mutagenic and/or cytotoxic, depending on how they interfere with the template function of the DNA during replication and transcription. DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy. All glycosylases share a common mode of action for damage recognition; they flip bases out of the DNA helix into a selective active site pocket, the architecture of which permits a sensitive detection of even minor base irregularities. Within the past few years, it has become clear that nature has exploited this ability to read the chemical structure of DNA bases for purposes other than canonical DNA repair. DNA glycosylases have been brought into context with molecular processes relating to innate and adaptive immunity as well as to the control of DNA methylation and epigenetic stability. Here, we summarize the key structural and mechanistic features of DNA glycosylases with a special focus on the mammalian enzymes, and then review the evidence for the newly emerging biological functions beyond the protection of genome integrity.

No abstract

Broken DNA ends are rejoined by non-homologous end-joining (NHEJ) pathways requiring the Ku proteins (Ku70, Ku80), DNA ligase IV and its associated protein Lif1/Xrcc4 (ref. 1). In mammalian meiotic cells, Ku protein levels are much lower than in somatic cells, apparently reducing the capacity of meiotic cells to carry out NHEJ and thereby promoting homologous recombination. In Saccharomyces cerevisiae, NHEJ is also downregulated in meiosis-competent MATa/MAT alpha diploid cells in comparison with diploids or haploids expressing only MATa or MAT alpha. Diploids expressing both MATa and MAT alpha show enhanced mitotic homologous recombination. Here we report that mating-type-dependent regulation of NHEJ in budding yeast is caused in part by transcriptional repression of both LIF1 and the gene NEJ1 (YLR265C)--identified from microarray screening of messenger RNAs. Deleting NEJ1 reduces NHEJ 100-fold in MATa or MAT alpha haploids. Constitutive expression of NEJ1, but not expression of LIF1, restores NHEJ in MATa/MAT alpha cells. Nej1 regulates the subcellular distribution of Lif1. A green fluorescent protein (GFP)-Lif1 fusion protein accumulates in the nucleus in cells expressing NEJ1 but is largely cytoplasmic when NEJ1 is repressed.

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...