5-Hydroxymethyl cytosine (5-hm-dC) is a minor DNA base; its presence in DNA strands was first observed in T-even bacteriophages (1). In such viruses, 5-hm-dC is often glycosylated, and this modified base protects phage DNA from cleavage by host restriction endonucleases after infection (2), and thus serves a direct epigenetic role in T-even phages. 5-hm-dC was first reported in mammalian systems in 1972, by Penn et al., who found relatively high levels of this modified base in DNA extracted from the brains of adult rats, mice and frogs (~ 15% of total cytosines) (3). In a follow-up study, Penn reported the observation of a highly statistically significant increase in 5-hm-dC in rat brain tissue as rats grew from newborn (~ 8% of total cytosines) to adult (~ 18% of total cytosines), and speculated that 5-hm-dC-containing DNA, or the base itself, might be implicated in the maintenance of steady-state neuronal activity, and possibly associated with synaptosomal mitochondria (4).
However, because the presence of 5-hm-dC in mammalian brain tissue could not be confirmed in other studies conducted around the same time, the topic languished for the next 30 years. Then, in 2009, Kriaucionis and Heintz (5) reported the presence of high levels of 5-hm-dC in Purkinje neurons from mouse brain tissue, with the 5-hm-dC specifically localized to CpG regions, thus both confirming the results of Penn et al.’s 1972 paper and expanding on it by definitively localizing 5-hm-dC to CpG regions of DNA, suggesting that this modified base plays an important epigenetic regulatory role in the central nervous system of mammals. Shortly thereafter, Tahiliani et al. (6) reported that the enzyme TET1 catalyzes the conversion of 5-methyl-dC to 5-hm-dC, both in vitro and in vivo, further strengthening the case for such a role.
However, because 5-hm-dC is also the oxidized form of 5-methyl-dC, it is possible that the former may sometimes simply be a damaged form of the latter, and thus could serve as a key intermediate in a possible DNA demethylation pathway involving interference in the ability of DNA methylases to perform maintainence methylation of dsDNA. Since the presence of 5-methyl-dC (in CpG islands) is important for suppressing gene expression in genomic DNA, conversion of 5-methyl-dC to 5-hm-dC via oxidative damage could lead to inappropriate activation of particular genes, which could contribute to aging, or the development of cancer (7).
The availability of 5-hm-dC as a phosphoramidite enables the incorporation of this modified base into synthetic oligonucleotides for use as research tools to help researchers definitively determine the role of this minor base in the biochemistry of brain and other tissues.
References
1. Wyatt, G.R.; Cohen, S.S. A new pyrimidine base from bacteriophage nucleic acids. Nature (London). (1952), 170: 1072-1073.
2. Wiberg, J.S. Amber Mutants of Bacteriophage T4 Defective in Deoxycytidine Diphosphatase and Deoxycytidine Triphosphatase—On the Role of 5-Hydroxymethylcytosine in Bacteriophage Deoxyribonucleic Acid. J. Biol. Chem. (1967), 242: 5824-5829.
3. Penn, N.W.; Suwalski, R.; O’Riley, C.; Bojanowski, K.; Yura, R. The Presence of 5-Hydroxymethylcytosine in Animal Deoxyribonucleic Acid.Biochem. J. (1972), 126: 781-790.
4. Penn, N.W. Modification of Brain Deoxyribonucleic Acid Base Content with Maturation in Normal and Malnourished Rats. Biochem. J. (1976), 155: 709-712.
5. Kriaucionis, S.; Heintz, N. The Nuclear Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain. Science (Published Online) (16 April, 2009), DOI: 10.1126/science.1169786: 1-3.
6. Tahiliani, M.; Koh, K.P.; Shen, Y.; Pastor, W.A.; Bandukwala, H.; Brudno, Y.; Agarwal, S.; Iyer, L.M.; Liu, D.; Aravind, L.; Rao, A. Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by MLL Partner TET1 Science (2000), 324: 930-935.
7. Ames, B.N. Endogenous DNA damage as related to cancer and aging. Mutat. Res. (1989), 214: 41-46.
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