DNA demethylation pathways: Additional players and regulators.

DNA demethylation can occur passively by "dilution" of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments

Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine

DNA cytosine-5 methylation is a well-studied epigenetic pathway implicated in gene expression control and disease pathogenesis. Different technologies have been developed to examine the distribution of 5-methylcytosine (5mC) in specific sequences of the genome. Recently, substantial amounts of 5-hydroxymethylcytosine (5hmC), most likely derived from enzymatic oxidation of 5mC by TET1, have been detected in certain mammalian tissues. Here, we have examined the ability of several commonly used DNA methylation profiling methods to distinguish between 5mC and 5hmC. We show that techniques based on sodium bisulfite treatment of DNA are incapable of distinguishing between the two modified bases. In contrast, techniques based on immunoprecipitation with anti-5mC antibody (methylated DNA immunoprecipitation, MeDIP) or those based on proteins that bind to methylated CpG sequences (e.g. methylated-CpG island recovery assay, MIRA) do not detect 5hmC and are specific for 5mC unless both modified bases occur in the same DNA fragment. We also report that several methyl-CpG binding proteins including MBD1, MBD2 and MBD4 do not bind to sequences containing 5hmC. Selective mapping of 5hmC will require the development of unique tools for the detection of this modified base

DNA glycosylases: in DNA repair and beyond

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