Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for whole-genome analysis of 5fC, based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

NEIL1 (Nei-like 1) is a DNA repair glycosylase guarding the mammalian genome against oxidized DNA bases. As the first enzymes in the base-excision repair pathway, glycosylases must recognize the cognate substrates and catalyze their excision. Here we present crystal structures of human NEIL1 bound to a range of duplex DNA. Together with computational and biochemical analyses, our results suggest that NEIL1 promotes tautomerization of thymine glycol (Tg)-a preferred substrate-for optimal binding in its active site. Moreover, this tautomerization event also facilitates NEIL1-catalyzed Tg excision. To our knowledge, the present example represents the first documented case of enzymepromoted tautomerization for efficient substrate recognition and catalysis in an enzyme-catalyzed reaction.base-excision repair | substrate recognition | enzyme catalysis | glycosylase | QM/MM D NA oxidation damage can be induced by both endogenous and environmental reactive oxygen species. Such oxidized DNA bases are primarily recognized and removed by the baseexcision repair (BER) pathway, which is initiated by a lesionspecific DNA glycosylase (1-4). Based on sequence homology and structural motifs, glycosylases that cleave oxidation damage are grouped into two families: the helix-hairpin-helix (HhH) family and the Fpg/Nei family (5, 6); the latter is named after the prototypical bacterial members formamidopyrimidine DNA glycosylase (Fpg) and endonuclease eight (Nei).NEIL1 (Nei-like 1) is one such Fpg/Nei family glycosylase that guards the mammalian genome against oxidation damage (7-10). NEIL1 is bifunctional in that it catalyzes both the hydrolysis of the N-glycosylic bond linking a base to a deoxyribose (glycosylase activity) and the subsequent cleavage of the DNA 3′ to the newly created apurinic/apyrimidinic site (lyase activity) (7-10). The N terminus contains the glycosylase domain of NEIL1, and the C terminus is intrinsically disordered (8,11). Whereas the C terminus is dispensable for both glycosylase and lyase activities in vitro, it interacts with many proteins in vivo and is required for efficient DNA repair activity inside the cells (12, 13). NEIL1 is also unique among the three human NEIL proteins in that it is increased in an S-phase-specific manner and carries out prereplicative repair of oxidized bases in the human genome (8,12). Moreover, increasing literature has further emphasized the importance of NEIL1's cellular repair activity, as NEIL1 deficiency has led to multiple abnormalities and is associated with severe human diseases, including cancer (14-19). Additionally, emerging evidence has also implicated a role of NEIL1 in active DNA demethylation (20)(21)(22).NEIL1 is capable of removing a wide array of oxidized pyrimidines and purines; representative substrates of extensive investigations include thymine glycol (Tg), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC), dihydrothymine (DHT), and dihydrouracil (DHU), as well as the formamidopyridines (FapyA and FapyG), spiroiminodihydantoin (Sp), and guanidinohydanto...

Uracil in DNA can be generated by cytosine deamination or dUMP misincorporation; however, its distribution in the human genome is poorly understood. Here we present a selective labeling and pull-down technology for genome-wide uracil profiling and identify thousands of uracil peaks in three different human cell lines. Surprisingly, uracil is highly enriched at the centromere of the human genome. Using mass spectrometry, we demonstrate that human centromeric DNA contains a higher level of uracil. We also directly verify the presence of uracil within two centromeric uracil peaks on chromosomes 6 and 11. Moreover, centromeric uracil is preferentially localized within the binding regions of the centromere-specific histone CENP-A and can be excised by human uracil-DNA glycosylase UNG. Collectively, our approaches allow comprehensive analysis of uracil in the human genome and provide robust tools for mapping and future functional studies of uracil in DNA.