Getting Vaccinia topoisomerase I to nick and covalently bind plasmid DNA - (Jul/27/2007 )

Hello,

We are trying to get commercially purchased DNA topoisomerase I from Vaccina (sold by Epicentre) to nick and covalently bind plasmid DNA. We are taking Qiagen miniprepped plasmid DNA (which contains two CCCTT recognition sequences 1125bp apart) and are treating it with DNA Topoisomerase I, Vaccinia under the reaction conditions specified by the supplier (http://www.epibio.com/pdftechlit/108pl076.pdf)

* 500ng-1ug miniprepped plasmid DNA
* 50 mM Tris-acetate (pH7.5)
* 100mM NaCl
* 2.5 mM MgCl2
* 0.1 mM EDTA
* 1 μL DNA topoisomerase I from Vaccinia

in a 50 uL volume.

We have also tried the the reaction conditions specified in the Shuman et al. papers. Namely,

* 50 mM Tris-HCl (pH 8.0)
* 100 mM NaCl
* 5 mM MgCl2
* 5 mM ATP
* 500ng-1ug miniprepped plasmid DNA
* 1 μL DNA topoisomerase I from Vaccinia

in a 20uL volume.

When we run treated and untreated plasmid on a 1.5% agarose gel, we see no difference between the two lanes (in either expt). If anyone has any idea why we might be having problems detecting nicking (do I need to run some other kind of gel?) or why the topoisomerase I might not be working? I ordered the enzyme fresh so I don't think it should be bad. I've also tried contacting Epicentre but no response so far.

Thanks in advance for any help anyone can offer.

Hmmm....
No difference was observed between the untreated supercoiled plasmid (raw) and the topoisomerase treated relaxed plasmid.

Is there a large difference between your open circular and the supercoiled DNA?
(Was a gel using raw plasmid run to check this? You should see some open circular, -although most of the DNA will be supercoiled.)

In fact, on that point I would recommend that you run a 1% agarose gel. 1.5% is far too dense a gel to clearly resolve whole plasmids of any size, and clearly tell the difference between supercoiled or open circular.

Is plasmid DNA clean? Since this is miniprep DNA, perhaps the plasmid too dirty to work with. Some enzymes are a bit more fussy then others when concerning their DNA. You could do a phenol/chloroform step to clean the DNA of proteins, followed by ethanol precipitation and 70% Ethanol wash to remove any residue phenol.

Another worry is the buffer that your plasmid was resuspended in. Did you use TE? If so, what was the volume of DNA that was added to the reaction mix? TE is composed of 1 mM EDTA, nearly 10x more EDTA then your buffer. And there is very little Mg ions in the buffer solution. Thus it is a possible danger that the EDTA might chelate and remove all free Mg ions in the buffer. Perhaps adding a little more Mg ion into the buffer might remedy the situation.

Best of luck

P.S: Probably a case of semantics, but you don't see nicking with Topoisomerase, you see relaxation of supercoils. Topoisomerase only breaks DNA strands, if the binding site is very close to the DNA end (<15bp) (linear DNA). In such a situation the short DNA end is released, and the Topo bound end can then capture another free DNA duplex (at high affinity) or water molecules/glycerol at low affinity (thus why Topo goes bad given enough time).

This is too specialized for me to answer. Was the tech service from the company any helpful to you?