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Old 04-02-2012, 12:54 PM   #1
pmiguel
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Lightbulb T4 ligases -- what do they ligate?

This post is a brief "journal club" sort of entry covering a topic that will be near and dear to our library prepping hearts...

What can be ligated?

Ligases are used to join polynucleotide strands. Three ligases derived from phage T4 have differing activities, depending on the nature of the strands being joined. There are two types of strands that can be joined: RNA or DNA. Further there are 3 components to a nick that is sealed (or not sealed) by a ligase. The 3' -OH donor, the 5' phosphate donor and the bottom or "template" strand holding the top strands flanking the nick adjacent to each other. Bullard and Bowater[1] assayed the effectiveness of all three T4 Ligases (T4 DNA Ligase, T4 RNA Ligase1 and T4 RNA Ligase2) for joining all eight types of nicks. Their results are presented below. Note that "0.001" is used where the authors used ">0.001". Also note the data are ploted on a base 2 log scale.




What constitutes "RNA" vs. "DNA"


Ignoring the "U" vs. "T" base difference, RNA has 2' hydroxylated ribose, but DNA is "deoxy" at that position. T4 RNA Ligase 2 appears to make the distinction among substrates based on the 2' carbon's hydroxylation status. For the 3' polynucleotide, only the final 2 bases need 2' -OH groups for T4 RNA Ligase 2 to function. The penultimate nucleoside sugar 2' -OH is needed for ligase to bind the nick, the ultimate to enhance 30-50x the efficiency of the join[2].

Interesting Results

Those who have long used T4 DNA ligase in cloning experiments may be surprised to learn that it has strong RNA-DNA nick joining ability if the template strand is DNA and the 5' phosphate-donating strand is DNA. Plasmid minipreps can easily contain 90% RNA. Not hard to imagine RNA interfering with cloning ligations under certain circumstances.

To a first approximation, T4 RNA ligase 2 will ligate the 3' -OH of an RNA strand to the 5' phosphate of DNA or RNA whether the bottom strand is DNA or RNA.


1. Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochemical Journal 398: 135-144.
2. Nandakumar J, Shuman S (2004) How an RNA Ligase Discriminates RNA versus DNA Damage. Molecular Cell 16: 211-221.

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Old 04-19-2012, 07:52 PM   #2
msheldon
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very nice piece of data. what is the r-> r/d part mean?
thanks
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Old 04-20-2012, 05:20 AM   #3
pmiguel
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Basically it is of the form A -> B/C. A->B denotes the molecule types of the oligos flanking the nick. That is, the oligos to be joined by the ligase. The /C denotes the type of oligo on the bottom strand. I guess it would be better as:

r -> d
-------
d

"r" denotes "RNA" and "d" denotes "DNA". The "->" is meant to denote 3' to 5'. So "A" is providing the 3' hydroxyl end and the "B" is providing the 5' phosphate end to be ligated.

So the chart depicts data from the referenced Bullard and Bowater paper where they determined the ability of the three T4 ligases to close a nick. Which might be a good proxy for their ability to ligate DNAs and/or RNAs together in a limited double-stranded context.

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Old 07-02-2012, 11:32 PM   #4
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Thank you so much for sharing this, Phillip. Just the info I had been looking for.

Do you know, or could reason, whether a phosphorothioate linkage between the last two 3' bases (near the nick site) in the RNA of R -> R/D would strongly affect the ligation by T4Rnl2?
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Old 07-03-2012, 09:19 AM   #5
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No idea.

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