I'm finally digging in on the grant proposal planning, with the goal of having a presentable draft before Christmas. A colleague in the US has agreed to critique it for me and I want to give him lots of time, and myself lots of time to fix it up once he's done.
I think I'm going to propose to test my hypothesis that DNA forms kinks at the USS during uptake.
Because the need to invoke kinks arises because cells can take up circular DNA molecules, I wonder if we should replicate the old experiments showing that closed circular molecules are efficiently taken up. There are lots of papers where circular plasmids were transformed into competent cells, but in these there was no analysis to rule out effects of nicked or linear plasmids contaminating the preparation. But I found the best paper, and the data looks quite solid. They used a preparation of closed circular molecules that gave a ladder of bands in gels, indicative of different extents of supercoiling, and the ladder was preserved in the DNA they later reextracted from cells that had taken the plasmids up. This confirms that the circular molecules were taken up.
It's possible that the kink arises because the DNA is nicked during uptake (it's easy to bend DNA sharply at a nick), but if so the nick must be resealed without changing the degree of supercoiling. This would require a specialized nicking enzyme for which we have no evidence. However we do have evidence of a ligase in the periplasm that could reseal nicks, though no idea why this would be beneficial.
So how does DNA form kinks? This time my literature searching led me into an area I'd missed previously - the ability of very short fragments to be ligated into circles. Linear DNAs longer than 200bp are easily ligated because the molecules spontaneously bend in smooth curves that (sometimes) bring the ends together. But shorter molecules (e.g. 100bp) were thought to not do this because their smooth curves would be too short - they'd make semicircles with the ends far apart instead of complete circles.
However Jon Widom's lab showed a few years ago that 100bp fragments form circles much more efficiently than anyone had expected, and this has directed attention to how spontaneous fluctuations in DNA structure cause transient kinking. One way this can happen is by formation of one or more internal bubbles (unpaired bases), because it's the base pairing that gives DNA its stiffness. Kinks can also be promoted by DNA-binding proteins that not only undo base pairs but flip one base out of the double helix
One issue that I don't think has been resolved is the extent to which the fluctuations only happen with short fragments. The ends of any double-stranded DNA molecules are known to spontaneously 'breathe', unzipping the terminal base pairs and zipping them up again due to thermal noise. In very short fragments this could affect base pairing throughout the fragment, facilitating formation of internal bubbles and kinks.
Saffron - pricey condiment
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