For this project to work I don’t just need parM to form nice filaments. There is also the other interactions to consider. ParM needs to interact with the parR adaptor protein, and parR needs to interact specifically to the parC sequence.
There could be many possible ways in which any of these interactions could be abolished in the eukaryotic setting vs the endogenous prokaryotic setting where parMRC works well.
To test this I split the system into 3 plasmids. The two plasmids expressing parM or parR where grown in bacteria without Dam methyltransferase. The third plasmid, encoding the parC was grown in normal E coli so m6A methylation by Dam would be present.
The reason for doing this is that our lab has previously developed the m6A tracer. A fluorescent fusion protein that is specific to the m6A methylation. Hence a fluorescent readout using the m6A tracer (after transfecting these 3 plasmids) will only highlight the plasmids that have the parC sequence. After expression of the parMR proteins it would be easy to see if there is correlation between the GFP signal of parM and the fluorescence of the parC plasmid. 3 outcomes are possible:
- Correct function: parM filaments with decorated ends from the parC plasmid.
- Specificity without function: parM filaments correlate to parC plasmid but no evidence of separation function.
- No correlation: parM and parC show no correlation. Project is dead!
After the experiment… We see outcomes 1 and 2! 🙂
When the concentration of plasmid is low and they are in close proximity you can observer normal function. An artifact of transient plasmid transfection is the high local concentration of plasmid molecules. When this occurs, there is still correlation of signal of the parM with the parC plasmids but the parMRC complex is not capable of disentangling it (although occasionally an escaper plasmid will be seen).
So we can conclude that parMRC is functional in mammals, at least in an episomal cytoplasmic setting. The challenge next is to check if parMRC can still function with parC in the native chromatin context…