5-hydroxymethyluridine DNA Kinase (5-HMUDK) transfers the gamma phosphate from ATP to the

hydroxymethyl moiety of 5-hydroxymethyluridine in polymeric DNA.

The 5-hydroxymethyluridine DNA Kinase enzyme is isolated from a strain of E. coli which contains the

htdk gene from Pseudomonas aeruginosa bacteriophage M6.

1X T4 DNA Ligase Reaction Buffer 
Incubate at 37°C.

10X T4 DNA Ligase Reaction Buffer

1X T4 DNA Ligase Reaction Buffer 
50 mM Tris-HCl 
10 mM MgCl2 
1 mM ATP 
10 mM DTT 
(pH 7.5 @ 25°C)

One unit is defined as the amount of enzyme required to protect 1 µg of Bacillus subtilis bacteriophage

SP8 genomic DNA in 30 minutes at 37°C in a total reaction volume of 20 µl against subsequent cleavage

by NcoI-HF restriction endonuclease.

Unit Assay Conditions
1X T4 DNA Ligase Reaction Buffer, 1 µg of Bacillus subtilis bacteriophage SP8 genomic DNA in a total

reaction volume of 20 µl. The reaction mix is incubated for 30 minutes at 37°C and then incubated at 80°C

for 10 minutes. Confirmation of phosphorylation is determined by protection of subsequent cleavage by

NcoI-HF restriction endonuclease. The reaction mix is treated with 2 µl of 10X CutSmart Buffer and 20 units

of NcoI-HF restriction endonuclease. Incubation at 37°C for 30 minutes is followed by analysis on an

agarose gel.

80°C for 10 min.

1. Weigele, P.R., et al. (2017). FASEB. 31,