Flipping over a molecular mousetrap

It has been a long-standing puzzle that some scientists have spent their whole careers mulling over: why during DNA replication do certain bacteria use a small protein called Tus to bind to the last part of DNA to be copied in a way that stops the replisome when it faces in one direction, but not in the other.

A collaborative effort between the Australian National University (ANU), the University of Southampton and the University of North Dakota has found that E. coli uses a 'molecular mousetrap' to block replication at the non-permissive end, involving a nucleotide 'flipping' over and inserting itself into a pocket on the surface of the Tus protein. Tus is then locked onto the DNA strand, stopping the replisome in its tracks.

ANU Research School of Chemistry's Professor Nick Dixon said that during chromosome synthesis in E. coli, replication forks are blocked by Tus-bound termination, or Ter, sites on approach from one direction (nonpermissive) but not the other.

"You have these two replication forks moving in opposite directions," Dixon said. "For some reason there is an advantage to the bacterium to have these defined points. These termination sequences are oriented in one direction of one arm of the chromosome and one is oriented in the other direction. "The replisome can go through the forks when they are in one arm of the orientation but not in the other. From a scientific point of view the key problem has been how is it that you can block this gigantic machine in this polar way, especially since this is a monomeric protein that binds to a simple DNA sequence. "From E. coli's point of view for some reason it's important to do this."

Strand separation of a few nucleotides at the permissive end is sufficient to force rapid dissociation of Tus and allow fork progression, he said. At the other end, a stable locked complex is formed by the G-C(6) base pair.

"We did a simple experiment where we made forked DNA sequences, just as the replisome would produce. Then we asked how fast does the Tus protein come off. When we made forks at one end, sure enough it got kicked off, and very quickly.

"At the other end where it gets blocked, nothing much happened for a while and then we got to a new complex that was incredibly stable, almost rock stable. We think that as the replisome comes along and pulls strands apart, it pulls in a few base pairs to form a new complex.

"We traced this to just one nucleotide that is flipping and binding into a pocket on the surface of the protein. Then we went ahead and determined the crystal structure of it to see what it looks like and you find that the protein actually makes a base pair essentially; it forms hydrogen bonds just like a Watson and Crick base pair, in a hydrophobic pocket that it sits in.

"It's a very neat solution and, going back through the literature, it explains a lot. It just works beautifully. We call it a molecular mousetrap because when the Tus binds to the site it primes it for this to happen, and as the helicose comes along and disturbs the trap, it flips."

Dixon said this unusual but simple mechanism only happens in E. coli and a few related bacteria. "There's an alternate system that happens in gram positive bacteria and it works probably by a different mechanism, more like a repressor/operator interaction. There's controversy still about how that system works. And then there's a whole lot of bacteria that aren't thought to have a termination system but probably do.

"It's neat and simple but what we haven't done yet is show that it works in an in vivo situation. That's the next step we are taking."