Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Summary

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Abstract

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5' hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

Introduction

Polynucleotide kinases (PNK) play critical roles in many DNA and RNA processing pathways, such as DNA repair and ribosome assembly^1,2,3,4,5. These fundamental enzymes catalyze the transfer of the terminal (gamma) monophosphate from a nucleoside triphosphate (NTP, most often ATP) to the 5' hydroxyl end of a nucleotide substrate. One of the most well characterized PNKs is bacteriophage T4 PNK, which has broad substrate specificity and is heavily utilized by molecular biology labs for incorporating radioac....

Protocol

1. Preparation

1. Prepare buffer and reagents.
   1. Make 1x Reaction Buffer by combining 20 µL of 1 M Tris (pH = 8.0), 40 µL of 5 M sodium chloride, 2.5 µL of 2 M magnesium chloride, 100 µL of 50% (v/v) glycerol, and RNase-free water to reach a total volume of 1 mL.
   2. Make urea loading dye by combining 4.8 g of urea, 200 µL of 1 M Tris (pH = 8.0), 20 µL of 0.5 M EDTA (pH = 8.0), 0.5 mL of 1% (w/v) bromophenol blue, and RNase-free water to reach a total volume of 10 mL.
   3. Make 10x TBE buffer by combining 108 g of Tris base, 55 g of boric acid, 40 mL of 0.5 M EDTA (pH = 8.0), and RNase-free water to reach a....

Results

A successful representative denaturing gel of a titration of ATP with a fixed amount of Las1-Grc3 complex is shown in Figure 1. Addition of enzyme resulted in Las1-mediated RNA cleavage of the SC-ITS2 RNA substrate, leading to a defined RNA fragment (5-OH C2 RNA). Upon the addition of ATP, the C2 RNA fragment was phosphorylated by Grc3 PNK (5-P C2 RNA). In denaturing gels the phosphorylated RNA migrates faster than its unphosphorylated counterpart. As shown i.......

Discussion

Described is an assay to measure kinase activity of Grc3 PNK on fluorescently-labeled nucleotide substrates. This protocol can be applied to characterize other PNK enzymes by adapting the Reaction Buffer and oligonucleotide substrate. For instance, the protocol calls for a trace amount of EDTA. The addition of EDTA is beneficial for two reasons: First, this approach favors magnesium-bound Grc3 by preventing the enzyme from binding to trace amounts of contaminating metals in the mixture. Second, a small amount of EDTA inh.......

Materials

Name	Company	Catalog Number	Comments
0.4 mm 34-well comb	BioRad	1653848
0.4 mm spacer	BioRad	1653812
0.5 M EDTA ph 8.0	KD Medical	RGF-3130
1M Magnesium Chloride	KD Medical	CAC-5290
1M Tris pH 8.0	KD Medical	RGF-3360
40% Acrylamide/Bis Solution 29:1	BioRad	1610146
5M Sodium Chloride	KD Medical	RGF-3720
ammonium persulfate (APS)	BioRad	161-0700
ATP	Sigma	A2383-1G
boric acid	Sigma	B0394
bromophenol blue sodium salt	Sigma	B5525-5G
Glass Plates	Thomas Scientific	1188K51
Hoefer SQ3 Sequencer	Hoefer	N/A
Image J Software	N/A	N/A	https://imagej.nih.gov/ij/
Labeled RNA oligonucleotides	IDT	Custom Order
Pharmacia EPS 3500 Power Supply	Pharmacia	N/A
Steriflip 0. 22 um Filter	Millipore	5FCP00525
TEMED	BioRad	161-0800
tris base	Sigma	TRIS-RO
Typhoon FLA 9500 gel imager	GE Healthcare	N/A
Ultra Pure DEPC Water	Invitrogen	750023
Ultra Pure Glycerol	Invitrogen	19E1056865
urea	Fisher Chemical	U15-500