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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 (1). However, TDG was also found to interact with transcription factors (2,3), histone acetyltransferases (4) and de novo DNA methyltransferases (5,6), and it has been associated with DNA demethylation in gene promoters following activation of transcription (7-9), 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 denovo methylation. We thus conclude that TDG-dependent DNA repair has evolved to provide epigenetic stability in lineage committed cells., TDG is one of four enzymes with UDG activity in mammalian cells, but its biological function has remained enigmatic (10). We thus set out to generate and phenotypically investigate a [...]

Spontaneous hydrolytic deamination of DNA bases represents a considerable mutagenic threat to all organisms, particularly those living in extreme habitats. Cytosine is readily deaminated to uracil, which base pairs with adenine during replication, and most organisms encode at least one uracil DNA glycosylase (UDG) that removes this aberrant base from DNA with high efficiency. Adenine deaminates to hypoxanthine approximately 10-fold less efficiently, and its removal from DNA in vivo has to date been reported to be mediated solely by alkyladenine DNA glycosylase. We previously showed that UdgB from Pyrobaculum aerophilum, a hyperthermophilic crenarchaeon, can excise hypoxanthine from oligonucleotide substrates, but as this organism is not amenable to genetic manipulation, we were unable to ascertain that the enzyme also has this role in vivo. In the present study, we show that UdgB from Mycobacterium smegmatis protects this organism against mutagenesis associated with deamination of both cytosine and adenine. Together with Ung-type uracil glycosylase, M. smegmatis UdgB also helps attenuate the cytotoxicity of the antimicrobial agent 5-fluorouracil. doi: 10.1128/JB.00613-09

In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.

Methylating agents of SN1 type are widely used in cancer chemotherapy, but their mode of action is poorly understood. In particular, it is unclear how the primary cytotoxic lesion, O'-methylguanine ([sup.ME]G), causes cell death. One hypothesis stipulates that binding of mismatch repair (MMR) proteins to [sup.ME]G/T mispairs arising during DNA replication triggers cell- cycle arrest and cell death. An alternative hypothesis posits that [sup.ME]G cytotoxicity is linked to futile processing of [sup.ME]G-containing base pairs by the MMR system. In this study, we provide compelling genetic evidence in support of the latter hypothesis. Treatment of 4644 deletion mutants of Saccharomyces cerevisiae with the prototypic [S.sub.N]1-type methylating agent N-methyl N'-nitro-N-nitrosoguanidine (MNNG) identified MMR as the only pathway that sensitizes cells to MNNG. In contrast, homologous recombination (HR), postreplicative repair, DNA helicases, and chromatin maintenance factors protect yeast cells against the cytotoxicity of this chemical. Notably, DNA damage signaling proteins played a protective rather than sensitizing role in the MNNG response. Taken together, this evidence demonstrates that [sup.ME]G-containing lesions in yeast must be processed to be cytotoxic.

It has been found that hMSH3 and hMSG6 interact with proliferating cell nuclear antigen (PCNA). They also go with the antigen to replication foci. PCNA, already implicated in eukaryotic postreplicative mismatch correction, binds to the human mismatch binding factors HMutS(alpha) and HMutS(beta) through their hMSH6 and hMSH3 subunits, respectively. PCNA appears to have a role in repair initiation by guiding the mismatch repair proteins to free termini in the DNA strands that are newly replicated.

MBD4, a mammalian protein that contains a methyl-CpG binding domain, has been found to efficiently remove thymine or uracil from a mismatched CpG site in vitro. The methyl-CpG binding domain of MBD4 binds preferentially to m5Cpg.TpG mismatches. It is suggested that this enzyme could act to minimize mutation at methyl-CpG. It is known that CpG to TpG transitions play an important role in genetic disease and cancer, and the impact of MBD4 loss on mutation frequency and cancer predisposition should therefore be investigated.