Abstract
Abstract 5-hydroxymethylcytosine (5-hmC) is an enzymatic oxidative product of 5-methylcytosine (5-mC). Ten Eleven Translocation (TET) family of enzymes catalyze the conversion of 5-mC to 5-hmC. Phage encoded glucosyltransferases are known to glucosylate 5-hmC, which can be utilized to detect and analyze the 5-hmC an epigenetic mark in the mammalian epigenome. Here we have performed a detailed biochemical characterization and steady-state kinetic parameter analysis of T4 phage β-glucosyltransferase (β-GT). Recombinant β-GT glucosylates 5-hmC DNA in a non-processive manner and binding to either 5-hmC DNA or uridine diphosphoglucose (UDP-glucose) substrates is random, with both binary complexes being catalytically competent. Product inhibition studies with β-GT demonstrated that UDP is a competitive inhibitor to UDP-glucose and a mixed inhibitor to 5-hmC DNA. Similarly, the glucosylated-5-hmC (5-ghmC) DNA is a competitive inhibitor to 5-hmC DNA and mixed inhibitor to UDP-glucose. 5-hmC DNA binds ∼10 fold stronger to the β-GT enzyme when compared to its glucosylated product. The numbers of 5-hmC on target sequences influenced the turnover numbers for recombinant β-GT. Furthermore, we have utilized recombinant β-GT to estimate global 5-hmC content in a variety of genomic DNAs. Most of the genomic DNAs derived from vertebrate tissue/cell-lines contained 5-hmC. DNA from mouse, human and bovine brains displayed 0.5 to 0.9% of the total nucleotides as 5-hmC, which was higher compared to other tissues. A comparison between cancer and healthy tissue genomes suggested a lower percentage of 5-hmC in cancer, which may reflect the global hypomethylation of 5-mC observed during oncogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-381. doi:1538-7445.AM2012-LB-381
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