Biochemical characterization of recombinant β-glucosyltransferase and analysis of global 5-hydroxymethylcytosine in unique genomes.
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 β-glucosyltransferase (β-GT). Recombinant β-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 β-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 ~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 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.
New England Biolabs, Inc., Ipswich, Massachusetts 01938, United States.
Jolyon
J
Terragni
Jurate
J
Bitinaite
Yu
Y
Zheng
Sriharsa
S
Pradhan
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology; D002417:Cattle; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000821:virology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005260:Female; D018745:Genome, Plant / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D051379:Mice; D041681:NIH 3T3 Cells; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D029681:Arabidopsis Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D005964:Glucosyltransferases; C022699:sucrose synthase; C026585:DNA beta-glucosyltransferase; C059776:endodeoxyribonuclease MfeI; D015252:Deoxyribonucleases, Type II Site-Specific