| |
[摘要]：5-Hydroxymethylcytosine (5-hmC) is an enzymatic oxidative product of 5-methylcytosine (5-mC). The 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 as an epigenetic mark in the mammalian epigenome. Here we have performed a detailed biochemical characterization and steady-state kinetic parameter analysis of T4 phage beta-glucosyltransferase (beta-GT). Recombinant beta-GT glucosylates 5-hmC DNA in a nonprocessive 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 beta-GT demonstrated that UDP is a competitive inhibitor with respect to UDP-glucose and a mixed inhibitor with respect to 5-hmC DNA. Similarly, the glucosylated-5-hmC (5-ghmC) DNA is a competitive inhibitor with respect to 5-hmC DNA and mixed inhibitor with respect to UDP-glucose. 5-hmC DNA binds similar to 10 fold stronger to the beta-GT enzyme when compared to its glucosylated product. The numbers of 5-hmC on target sequences influenced the turnover numbers for recombinant beta-GT. Furthermore, we have utilized recombinant beta-GT to estimate global 5-hmC content in a variety of genomic DNAs. Most of the genomic DNAs derived from vertebrate tissue and cell lines contained 5-hmC. DNA from mouse, human, and bovine brains displayed 0.5-0.9% of the total nucleotides as 5-hmC, which was higher compared to the levels found in 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. |
|
