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DNA Replication Initiation
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0:08
Hi. My name is Steve Bell, and I'm a professor of biology at MIT and an investigator of the Howard Hughes Medical Institute. And what I'd like to tell you about now is the mechanisms controlling the initiation of DNA replication at chromosomes. This is a continuation of my previous discussion of the events that occur at the replication fork. Now, this process involves two key events. First, unwind part of the DNA to make single-stranded templates to assemble the replisome on.
0:40
And, secondly, you need to assemble two copies of the replisome, because all genomic or chromosomal origins of replication initiate DNA replication bidirectionally, meaning that they... the replisomes that are assembled move away in both directions from the starting site of replication. So, the start sites of DNA replication are called origins of replication, and this is where the first single-stranded DNA is formed and where the first DNA synthesis occurs.
1:09
They can fire with various so-called efficiencies, which is defined as the percentage of cell divisions in which an origin initiates DNA replication. Some origins have high efficiencies, others have lower, and I'll tell you more about that in a moment. Now, all the DNA that's replicated by the two replisomes that are formed at each origin of replication... and, a reminder, all chromosomal origins are bidirectional... that DNA is called a replicon. And this stems from a model proposed in 1963
1:40
by Jacob, Brenner, and Cuzin for the mechanism of initiation of bacterial replication. It's a very simple model. They proposed only two components: the replicator and the initiator. The replicator is the DNA sequences that are required to initiate DNA replication. The initiator is a protein that was proposed to bind to the replicator and trigger the initiation of DNA replication. Now, the replicator, although it typically overlaps the origin of replication, is not the same as that,
2:12
and it is instead all the sequences that are required. And this is sometimes a little bit difficult to understand, but think about it in terms of transcription, where the promoter is all the sequences required to correctly regulate initiation of transcription from the gene, but the transcriptional start site is a very specific sequence at which initiation actually occurs. In that analogy, the promoter is equivalent to the replicator, and the transcriptional start site
2:39
is equivalent to the origin of replication. So, this is what origins are like and what replicators are like... how are they distributed across the chromosomes? So, in E. coli, not surprisingly, where there's a single origin of replication, the origins are 100% efficient. If an origin doesn't fire, that means one of the two daughter cells is not getting a whole chromosome. This is different in eukaryotic cells, where there are many, many origins. What's found is that most origins
3:11
do not initiate 100% of the time, but that's okay, because when they don't initiate, the replication forks on either side will replicate through the region that they would have replicated otherwise, because there's lot of excess replication capacity. It's important to note that any origin that doesn't initiate but is replicated is inactivated. So, you never have an origin initiate after it's been replicated by an adjacent fork. Now, another interesting characteristic of eukaryotic replication
3:42
is that when origins initiate is characteristic of different origins. Some will initiate early during the S phase of the cell cycle, the time in the eukaryotic cell cycle when DNA replication occurs, others will replicate in the middle, and still others will replicate late in S phase. And it's sort of initially unclear why you'd want to distribute this. Why not replicate everything all at once? But it's more clear when you think about the fact that sometimes you'll have damage to your DNA
4:11
-- this could occur by a sudden change in your environment -- and if you do and you stop two forks, and they haven't replicated the intervening DNA, the only way to rescue that situation is to initiate replication between those two forks and create two new replisomes that can fill in that gap. And by reserving a subset of origins to initiate late in S phase, you will increase the likelihood that you can fix a gap that's caused by some sort of DNA damage and stalling of replication forks. Okay?
4:47
So, we've talked about origins, let's talk about how initiation occurs? So, we're going to start with the initiation of DNA replication in bacteria. So, this is an illustration of the E. coli origin of replication. It has 5 copies of the so-called 9-mer repeats, which are the binding sites for the bacterial initiator protein, which is called DnaA, and 3 copies of the 13-mer repeat, which is an AT-rich, easily unwound DNA sequence. Now, when the DnaA is present,
5:20
which is... and active in the cell, and this is a highly regulated step in the process in bacterial cells, it will bind to the 9-mer sequences. This is turn forms a helical filament, which distorts the DNA, causing the DNA it's bound to have positive superhelicity, and the adjacent DNA to, in a compensatory fashion, generate negative superhelicity, which favors unwinding of the adjacent 13-mer regions. Interestingly, the DnaA that forms this filament can actually extend that filament
5:57
by binding the single-stranded DNA rather than the double-stranded DNA, although it only does this on the top strand, as illustrated here, not on both strands. Now, at this point, we've accomplished one important event, which is to unwind the DNA at the origin, but we're clearly lacking much of the rest of the machinery, and the next thing we need to initiate replication is to load the replicative helicase... with DnaB helicase, the replicative helicase.
6:26
Now, it turns out that it doesn't arrive at the origin on its own; it's bound to another protein, DnaC, which is a so-called helicase loader, and it has many important properties that make it ideal for loading the helicase on the DNA. So, one property is when DnaC is bound to DnaB, it inactivates its helicase activity, preventing it from acting at non-origin regions of the genome. Secondly, DnaC causes the DnaB ring to crack open such that it has enough room to fit a single-stranded DNA
7:04
so that it can gain access into the central channel of the hexameric ring structure. The third important property is that DnaC has affinity for DnaA, which recruits the DnaB-C complex to the single-stranded DNA and allows it to now encircle the DNA, so it binds such that it will now encircle the single-stranded DNA. Now importantly not only does the DnaB-C complex load on the top strand that has DnaA associated with it, but it also is loaded on the bottom strand, but in the opposite orientation,
7:40
so that it will leave the replication... the origin in the opposite direction. So, you can see that on the top it's gonna move to the left, on the bottom it's gonna move to the right. Now, we don't understand in detail how that second DnaB-C complex is recruited, although there are theories that suggest that DnaA, which also has affinity for DnaB, may use that interaction to recruit the second B-C complex in the opposite orientation. Now, at this point,
8:12
you'll remember that the B-C complex is inactive, so the helicase is not active yet. Nevertheless, you'll recall that the interaction between the DNA primase and the helicase stimulates the primase to synthesize new primers, and that's the next step of this process. The DNA primase is recruited, binds to the helicase, and synthesizes a primer, and something about this process causes the DnaC subunits to release. Again, we don't understand that in detail,
8:43
but that's clearly the step at which this occurs, and now you have active helicases, which can move to the right and the left -- top to the left, bottom to the right -- to unwind more DNA, so that you get an extended unwound region. Now, at this point, we have two primer-template junctions that can be recognized by DNA polymerase III holoenzyme, load sliding clamps, and then the sliding clamps can be recognized by one of the three DNA polymerase III enzymes.
9:12
It will then initiate leading strand synthesis. Then, at some rate determined by the affinity between the helicase and the primase, a second primer will be synthesized. That will be recognized by the clamp loader and a second polymerase subunit, and then you have initiated both leading and lagging strand synthesis, and this is really the starting point that we discussed in terms of the function of the replisome during elongation, so you know what happens from here.
9:40
Now, I want to make a couple other points at this point. You'll note that there was only a single sequence-specific DNA binding protein involved in this process. Most of the process is driven by either protein-protein interactions or non-specific or structure-specific protein-DNA interactions. So, for example, the recognition of the primer-template junction is sequence-non-specific, but very specific for the particular structure, that of a primer with the 3' OH available
10:11
and an adjacent single-stranded region for the DNA polymerase to be engaged with. Okay? And that's true also for the primase interacting with the helicase and the DNA polymerase III holoenzyme interacting with the helicase as well. So, this is built not by a series of sequence-specific interactions, but by a series of protein-protein-specific interactions or structure-specific interactions. Okay, so that's the initiation in bacteria. I want to turn my attention, now,
10:43
to initiation in eukaryotic cells, which is substantially more complex. So, this is just an overview of the events of eukaryotic DNA replication, and I first want to discuss the overview, then I'm going to describe how these events are regulated, and then I'm going to take you through the detailed steps of this initiation event. So, the first step in this process occurs not during the S phase of the cell cycle, when DNA synthesis occurs, but during the G1 phase of the cell cycle,
11:13
so the preparations for replication occur well before any nucleotide is added to any DNA strand. During this helicase loading process in G1, the replicative helicase, this Mcm2-7 complex, is assembled around the DNA at each origin. In fact, this marks all potential origins in cells and is frequently referred to as origin licensing or sometimes pre-replicative complex formation. Three additional proteins are required to accomplish this. In addition to the helicase, the origin recognition complex,
11:51
Cdc6, and Cdt1, and I'll have much more to say about them in a few minutes. As the cells go from the G1 phase to the S phase of the cell cycle, the action of two kinases, the S phase cyclin-dependent kinase, and the Dbf4-dependent kinase, or DDK, leads to the accumulation of a number of different proteins onto the loaded replicative helicase, because what I didn't tell you is that when it's initially loaded it's surrounding double-stranded DNA and it's completely inactive, okay?
12:25
So, it's important that it's inactive at that point and you'll see why. Now, these associated proteins include two proteins that I discussed before, Cdc45 and GINS, and they come together to activate the helicase and form the active replicative helicase, the CMG complex. Now, the single-stranded DNA that's formed at this point allows the recruitment of the rest of the DNA synthetic machinery and assembly of a pair of bidirectional replisomes. Now, what's important about this process
12:57
is that the helicase loading step is separated from the helicase activation step. And this is critical for one very important attribute of eukaryotic DNA replication, which is that the genome is replicated exactly once per cell cycle. You never want an origin to initiate more than once per cell cycle. So, I want to talk about how the cell ensures that over the next couple of slides. So, during the G1 phase of the cell cycle, there is low levels of an enzyme called cyclin-dependent kinase.
13:28
This is the key enzyme that drives cell cycle progression. During G1, there's only G1 cyclin-dependent kinases -- no S phase cyclin-dependent kinases -- and in general their activity is much lower. Under these conditions, you can assemble or load new replicative helicases at the origins of replication, but you cannot activate them, because, as I told you in the previous slide, you require S phase CDK activity for this. When you transition into the S phase of the cell cycle, S phase CDKs,
14:01
and in fact CDKs in general, become much more represented, much higher activity, and they have two consequences. One is that they trigger initiation of any loaded helicases, as I described in the previous slide. The second important consequence is that they also target three of the four proteins involved in helicase loading -- the Mcm2-7 complex, ORC, and Cdc6 -- and each of these phosphorylation events inhibits helicase loading. I'm going to describe that in detail in a moment,
14:38
but let me just explain the logic of this system first. So, you can see that in this system there's only one chance, during G1, to load a replicative helicase per cell cycle, and there's only one chance, during S phase, typically, but really the whole period from S, G2, and M, where you could actually activate a helicase. In practice, this all occurs during S phase for a variety of reasons that I won't go into, but what this means is you basically can only initiate replication from an origin
15:07
once per cell cycle. And this system works rather you have 10 origins, 1000 origins, or 30000 origins. It's going to work equally well. And so it's very able to accommodate many different genome sizes. Now, you might wonder what happens at the G1-S and M-G1 transitions. That's really a dangerous period where you want to stop helicase loading before you start helicase activation. If there was some gradual transition, you could have a brief period where they both could occur at the same time,
15:38
and that could allow re-replication to occur. However, it turns out the cell cycle has come up with mechanisms, and I won't go through these in detail, that turn off helicase loading before you turn on helicase activation at the G1-S transition, and similarly you turn off activation before you turn on helicase loading at the M-G1 transition. So, at each of these key transitions, you have a little window where neither of them can occur. Okay? And I should just emphasize that this transition
16:11
at the M to G1 is exactly where cell division occurs, so you can now start the process again, because you now have two cells, each of which has a complete complement of the genome in it and is ready to initiate a new round of cell division and therefore DNA synthesis. Now, how does S CDK control the helicase loading proteins? We know that best in the budding yeast S. cerevisiae, where we know that, for example, cyclin-CDKs phosphorylate the Mcm proteins, which causes them to be
16:45
exported out of the nucleus. Now, this may be confusing to you because obviously there are many Mcm proteins that are loaded onto the DNA that you don't want to leave the nucleus, and the important point here is that only non-DNA associated Mcms are exported. So, as soon as they finish their function, they're exported to the cytoplasm, but if they're waiting at an origin, loaded, to be activated, they are not exported out by CDK phosphorylation. Cdc6 is also phosphorylated by CDK,
17:16
and that phosphorylation leads to its recognition by a protein complex called SCF, which is a ubiquitin ligase, which targets Cdc6 with multiple modifications of ubiquitin, which in turn causes Cdc6 to be recognized by the proteasome and degraded. So, except for in G1, Cdc6 is consistently being degraded and therefore cannot function. Finally, ORC is also phosphorylated, and in this case we know less about this, but it's prob... most likely an event that inhibits the interaction of
17:50
one of the other helicase loading proteins with ORC. Okay. So, I've told you about the regulation of this event, let's talk about how it actually occurs. So, this is just an illustration of the events of helicase loading. We know that the first step in this process is the recognition of the origin DNA by the origin recognition complex, or what I'll call ORC from now on. ORC then recruits Cdc6 as cells enter into the G1 phase of the cell cycle, when it's newly synthesized,
18:19
and then the ORC-Cdc6 complex can recruit a complex between the Mcm2-7 helicase and Cdt1. And this forms this initial complex called the OCCM, for ORC, Cdc6, Cdt1, Mcm complex. And I want to point out that, at this point, it's the C-terminal end of the Mcm complexes, right here, that's interacting with ORC. The ring is still open at the this point, but they're bound to adjacent pieces of DNA, making it very clear how you recruit the first Mcm to the DNA. Now, the next step after this
18:56
requires ATP hydrolysis, and involves a number of steps that I'll talk about in more detail in my second part of my presentation. But the end result is that you end up with a double hexamer of Mcms on the DNA, with then interacting via their N-terminal regions to form a head-to-head double hexamer of the proteins on the DNA, and this is really the first marker of bidirectional initiation, because they are now poised to leave the origin in opposite directions. So, this happens during G1.
19:30
Again, the helicases are inactive at this point, and what happens next is you transition into S phase and you undergo helicase activation. Now, this is an overview of this process, and I'm going to go over this first and then I'm going to go over the steps in detail. So, the first event is... it requires the DDK kinase and it results in the recruitment of one of the two activators, the Cdc45 protein. Then, S-CDK can act and this recruits the other activator, the GINS complex,
20:04
as well as Pol ε and several other proteins. At this point, you have two CMGs that have formed on the template, but interestingly they are not yet active. It requires the activity of a third protein, the Mcm10 protein, to actually trigger DNA unwinding and CMG activation. And this also requires the association of the eukaryotic single-stranded binding protein RPA. Finally, once the single-stranded DNA is formed, the remaining DNA polymerases, as well as other auxiliary proteins,
20:39
are recruited to form the replisome. Now, this is a lot to look at, so I want to break this down into individual steps so you can understand how this occurs in more detail. So, here's our loaded double hexamer, and it's ready to be activated, just entering into S phase. So, the first step is the DDK recognition of the N-terminal region of several of the Mcm subunits. It phosphorylates these subunits and that creates a binding site for a second protein called Sld3,
21:13
which in turn allows the recruitment of one of the two activating proteins, Cdc45. Now, I've shown this for the right hexamer, but of course this also has to happen for the left hexamer as well. Okay, so now we have the first activator present, and that's a DDK, or Dbf4-dependent kinase-dependent event. The next step absolutely requires the S phase cyclin-dependent kinase, and what it does it to phosphorylate first Sld3 and then, second, it phosphorylates... not necessarily in that order,
21:48
but it also phosphorylates Sld2. Now, these phosphorylation events cause the recruitment of a variety of additional proteins. Most notably, Sld2 is recognized by a protein called Dpb11, and it does this using a pair of BRCT repeat motifs, which recognize, in a phosphorylation-specific manner, a peptide on Sld2. This phosphorylation event also causes the recruitment of Pol ε and the GINS protein into a larger complex that's called the pre-landing complex, or pre-LC.
22:25
Now, this complex is recruited as a whole to the inactive, ready-to-be-activated helicase, using the interaction of the Dpb11 protein with a different phosphorylation... phosphorylated peptide, okay? And this, again, is mediated by a separate pair of BRCT motifs, which recognize, in a phosphorylation-specific manner, peptides within Sld3. So, at this point, we've recruited both Cdc45 and GINS, and so we have a CMG complex present, okay, but it's not active yet.
23:05
And of course we also have to do this for the other hexamer. Okay, so what happens next is really a point of major investigation in the field, but there are a number of things we know must happen. So, one of these is that several of the proteins leave the replisome. This includes Sld2, Sld3, and Dpb11. So, we have our CMG complex, and this is, again, the active replicative helicase, but it's not active yet because in order for the first unwinding of DNA to occur the Mcm10 protein has to act.
23:40
And we know that this actually directly stimulates helicase activity, and that it can cause a number of changes in order to cause initial unwinding. Exactly the order of these changes, and whether Mcm10 or earlier events may stimulate them, is unknown. We only know that you don't see single-stranded DNA until Mcm10 arrives. But this structure, which has now got RPA binding to the single-stranded regions, has a number of dramatic changes versus the previous structure. So, of course the origin DNA
24:15
had to be melted in order to create single-stranded DNA. In addition, the non-translocating strand had to be exported from the central channel. Remember, the Mcms were around double-stranded DNA initially, however, after this transition the right helicase is going to have one single-strand present in it, and the left helicase is going to have a different single-strand in it. So, that means these Mcm complexes have to open, eject one single-strand, and then close again around another.
24:47
And, finally, you'll recall that the helicases are in this tight, head-to-head double hexamer, initially, and they have to separate in this process. So, I've listed these in an order, but we don't know if this is actually the order they occur in -- that's an area of active investigation. Okay. So, at this point we know have the single-stranded DNA necessary for priming, and what happens is that the rest of the DNA synthetic machinery is recruited. In particular, Pol α-primase,
25:16
is recruited by interactions of Ctf4 with the GINS complex, and also with Pol α. This allows the synthesis of the first RNA primers and then the extension of those primers by Pol α in the form of DNA. Those primers can then be taken over, either by Pol δ or by Pol ε, depending on whether they're on the leading or the lagging strand, and you now have an active replisome. So, there's lots more to be understood about this process -- it's much less well-understood
25:52
what the architecture of this complex is -- and there's a lot of investigation going on at this point to understand that. For example, I've illustrated Pol δ being at the site of replication, but a lot of evidence suggests that it's actually after... it's occurring sort of after the fork has gone by. So, it's doing its job, but somewhat separately from the job of the rest of the polymerases. I'll also point out that I haven't shown either the sliding clamp loader, RF-C, or sliding clamps.
26:20
They would both be acting, but, again, they're not part of the actual replication fork. So, I hope you've learned a lot about how replication works, here, and if you stay tuned I'll tell you much more detail about how the helicases are loading, and what we've learned by using single-molecule biochemistry to investigate this process. So, I'd like to thank Sera Thornton, who did all the animations in this part of my presentation. These were done as part of the development of a course called 728x,
26:51
which is available on the MITx and edX platforms, and is of course focused on molecular biology, not just DNA replication but all of the central dogma. I'd also like to thank my funding resources, the Howard Hughes Medical Institute, as well as the National Institute of General Medical Sciences.
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