http://www.wehi.edu.au/education/wehi-tv/dna/replication.html

{It is best to wait for all four of them to load and watch simultaneously PS How Do I embed the damn things?}

http://www.wehi.edu.au/education/wehi-tv/dna/movies/Replication_F.mov

http://www.wehi.edu.au/education/wehi-tv/dna/movies/Replication_AB.mov

http://www.wehi.edu.au/education/wehi-tv/dna/movies/Replication_BL.mov

http://www.wehi.edu.au/education/wehi-tv/dna/movies/Replication_BR.mov

Ok, Just so everyone knows what is going on, this is a Virtual Rendering of DNA replication. Some of the mechanisms have been left out because if they were put in there, no one would be able to tell what was going on. The video is showing us what DNA replication would look like if a cell had no water and one second lasted one minute.

Your basic rundown of DNA replication goes like this. DNA is a double helix structure, made up of nucleotides. A nucleotide consists of a ring-structured sugar, a phosphate, and a chemical called a base, of which there are four types, which hold the information in DNA, whilst the sugar and phosphate merely provide the backbone. Hence, a piece of ssDNA (single strand DNA) appears to be shaped like a ladder which has been cut in half along its long axis. The rungs are complementary, so the bases only recognize a specific counterpart and bind to it very tightly by forming strong hydrogen bonds (which are normally very weak, but their strength is greatly exacerbated by the fact that the DNA molecules is millions of bases in length). The base called Adenine only recognizes Thymine, and cytosine only recognizes guanine. In this way, DNA can be assembled by templated polymerization, ie, on a single strand of DNA, ie half a ladder, the other half can be synthesized by that the nucleotides will slot into correct place on the DNA strand because they only recognize one counterpart. In polymerization, the monomers are strung together by a reactive bond. In this case, a nucleotide triphostate (ATP, GTP, CTP or TTP) is hydrolyzed (split by water) such that the pyrophosphate (two of the phosphates) are displaced and a highly reactive phosphate bond is left on the nucleotide, via which it joins to its fellow nucleotide already on the strand. In this way, each incoming nucleotide carries its own reactive bond to join the growing chain. This is called tails polymerization.

Now, DNA replication works like this. The double-helix peels apart, so the single strands are exposed, and free nucleotides can then be polymerized on the two template strands, and so, when all is said and done, we will have two exact copies of what we just had. The problem is that, for reasons that I don't need to go through now, DNA can only polymerize in one direction, from the 5' end (the phosphate) to the 3' end (the sugar slot of the next nucleotide). Now, the two strands are asymmetric. THis means that they run antiparallel to each other. One strand runs in the 5' to 3' direction whilst its complement runs 3' to 5'. Without this, the DNA could never constitute itself into the double helix it has become famous for.

This means that since DNA polymerization can only occur from 5' to 3' one of the strands has to synthesize backwards, which entails that it assemble in the opposite direction to the strand it is trying to synthesize on. The result is that instead of having a continuous flow of nucleotides being stitched into the assembling strand, like what occurs on the leading strand, the lagging strand is synthesized by a constant discontinous switch-stitching by synthesizing small pieces in the opposing direction, called Okazaki fragments.

So, as you can see, the Double helix enters that large blue thing where it is peeled apart and forked into two different enzyme complexes. That blue thing is an enzyme called DNA helicase and it runs along the strand forcing it open to expose the bases. That purple enzyme is DNA polymerase and it catalyzes the addition of free nucleotides. Now, the strand on the left is running backwards because it has to synthesize Okazaki fragments discontinuously. That is why the polymerase keeps shifting. Polymerase cannot begin a new strand de novo, it has to have a primer, which are backstitched into the lagging strand by RNA primase, from which the polymerase catalyzes polymerization in the opposing direction, once the discontinuous stitching runs into the next primer (which it will because it is going backwards) the polymerase disassociates from the complex because the stop in motion causes the ring-clamp loader complex, which holds the polymerase to the DNA, to disassociate. The RNA primase can synthesize the addition of two nucleotides onto a template strand without adding them to preexisting nucleotides, so it can make a new strand de novo. It uses RNA, slightly different from DNA but still readable by it, and the RNA is then erased once the DNA polymerase has started adding more nucleotides from the end of the RNA primer. The polymerase absolutely requires a 3' end OH bond to attach the next nucleotide, it can never add two together de novo, it just doesn't catalyze that reaction (there is a good reason for this, it has to do with the error correction mechanism in DNA polymerase). THe DNA polymerase is then added to the DNA by means of the just-discussed clamp, which is assembled from two pieces and loaded onto the strand by a clamp loader protein, which is then displaced by the polymerase. Without the clamp ring, the DNA polymerase would fall off the DNA strand very quickly. It doesn't bind to it very tightly. This is useful because once the whole mechanism stops moving (like when the machine runs into the next section and therefore has to stop), the clamp diassociates and so the polymerase falls off. This mechanism ensures it does not overshoot its mark.

That is what those two semicircular pieces which keep flying on and off are, the clamps. If you look closely, you'll see that when the Clamp-Loader-Polymerase Complex actually makes that swift rotating motion before everything flies off, the synthesis runs into a freshly synthesized double helix, which again is because the lagging strand is stitching bachwards. The video marked "Front" is by far the best way to see this.

All of the proteins in question are held together in a multienzyme complex called a fork, called so, because it is indeed a fork. In it, you can see that the central fork is polymerizing an Okazaki fragment very clearly. A small primase quickly forms a small RNA primer and then diassociates, at which point the clamp is loaded onto the DNA by the loader, which causes it to fold over, where the loader protein dissasociates from the complex (although it stays in proximity for the next strand) and the polymerase is loaded on (the purple hand-shaped protein). At that same moment, you should be able to see a freshly completely piece of strand disassociate from the polymerase, because it ran into the RNA primer of the next section on the lagging strand. This causes the whole protein machinery to dissasociate from the DNA strand, so it can reassamble for the next piece.

On the other hand, if you watch the leading strand, you'll see that it is continuous, ie the ring-clamp-polymerase never disassociate or shift because they don't need to, the whole strand can be run through it without incident. The reason the other strand is always shifting, torquing and the pieces are flying off is because the DNA is being stitched in backwards. So, after being peeled apart, an RNA primase needs to add a short primer strand so the polymerase can begin, once it does that, it will continue for about 2000 base pairs along the lagging strand (the length of the average Okazaki fragment), and then strikes the next already done strand, everything flies off and reassociates for the next fresh piece. THe RNA primer is erased very quickly and then there is a left over gap in the DNA which is filled by an enzyme called DNA ligase which adds a reactive bond to the broken strand which is quickly cut and so the broken nucleotides join together.

Another thing to watch for is that the RNA primase continually flies on and off the helicase, to form a complex called a primosome. The primosome moves across the lagging strand synthesizing RNA primers across Okazaki fragments as it goes. This allows DNA polymerase to fill in the gaps, the RNA primer to erase and the ligase to seal the nick. This is the reason that the lagging strand keeps "folding back", as you can see. It allows the two polymerases to be bound in a multienzyme complex with the helicase. If this were not the case, the polymerase-clamp complex would run along the DNA in front of the helicase of its own accord, and then would have to keep waiting for the primosome to synthesize more RNA on the lagging strand. So, forcing the whole thing to act as a single piece of machinery makes the whole process vastly more efficient, which is the reason that the lagging strand keeps folding inwards on itself. You won't be able to see the repair enzymes that erase and reseal the RNA primers and plug the nicks, as those operate behind the replication machine in its wake, and the addition of the extra proteins would make the video too confusing to watch properly, although they really should be there.

Completely counterintuively, although you can see the DNA appearing to be "threaded through" the protein complex, it is actually the other way around. It isn't the DNA that is moving. It is the protein complex that is running through it (This is obvious when we think about it, there would be nothing to drive the motion the other way). Also, whilst I would estimate that the DNA in these videos is being synthesized at perhaps 20 base pairs per second, real cellular machinery can vary from 1000 to 10,000 nucleotides per second. This is understandable given that "speed" has a very different meaning at the molecular level. Normally, that protein complex would, like most proteins, be tumbling about its main axis...about a million times per second. Also, you would be able to see constant streams of hundreds of thousands of base pairs so fast that they would completely blank the screen. If all of this was shown on the video, the whole thing would be a continuous blur, a blank screen to our eyes.

Some things are missing from this model in the video. Normally, a modular protein called an SSB or a helix destabilizer is holding the DNA rather the way that coating a malleable wire in plastic keeps it rigid, which ensures the DNA doesn't kink, knot or start reacting with the bases on its own strand to form hairpins which interfers with the process. However, if the video showed this, we would not be able to see the replication taking place, so it has been ommited. There are a vast number of other mechanisms that are not being shown for clarity purposes, otherwise the whole thing would just look like a confusing blur of proteins flying on and off another blob of proteins. For example, an enzyme called 3' to 5' exonuclease destroys mutated tautomeric forms of DNA that occasionally bind to the template by accident. Then there are topoisomerases which keep the DNA from tangling once it turns to form a double helix again. In short, the only thing we are being shown in this video is the replication machine, which constitutes only part of the DNA replication process.

I want to stress that since so many things are missing from the video, the replication would never work. The helicase appears to be unwinding the DNA on its own. In reality it could never do this. The helicase can pry apart the strands once they've straightened, but whilst in their double-helical topology, they are extremely tightly wound and therefore incredibly strong and with a very high structural integrity. What the video is displaying you would be akin to trying to undo the pressurized bolts on a nuclear submarine using a paperclip. What topoisomerase does is that it makes a temporary break in the DNA backbone allowing part of it to freely rotate, this will always force the change in shape in the direction that relieves the immense torque generated by the tight double helical configuration, once it has been straightened out, the topoisomerase reconstitutes the structural integrity of the DNA by adding the phosphate back. Whilst you can see the tightly wound double helical DNA being moved along the helicase, in reality the topoisomerase moves in front of it temporarily breaking the structural integrity (it is best to view this using the Back Left video) you can see that the DNA being funnelled through the helicase is still tightly wound. But as I said above, the helicase can only peel apart the DNA once it has been unwound, whilst wound, the enzyme is useless. We should see both (a) a clamped topoisomerase breaking the strand, allowing it to unwind and relieve the extreme pressure and (b) straight, unwound DNA being pried by the helicase.

Also note that the video says that this is a remix of Eukaryotic and Prokaryotic replisomes. This is correct. The replisome in this fork appears to have what we call an alpha polymerase complex, as well as two delta polymerase complexes. This is not present in Bacterial forks. You can see that this appears to be a triple-pronged fork. The extra prong comes from the fact that Eukaryotic mechanisms have three polymerases, although we don't actually know why this is. 

However, if this is a model of Eukaroytic replication, it is horribly inaccurate. Eukaryotic replication explicitly takes place on the surface of a low-level structure of chromosomal organization known as a nucleosome. A giant protein drum-like shape made of eight subunits around which DNA is wrapped. We should definitely be able to see the replication fork moving across the DNA on the nucleosome, but in the video the DNA appears to be free in the cell. This is how it is organized in bacteria. So, we can see that the video shows us a model that does not actually exist in real life, but is a helpful illustration of concepts without adding all the confusing parts in which would make it nearly impossible to see what was going on.

But it's still a pretty damn good illustration.