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Origins of Replication · Acedra
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Origins of Replication
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0:03
video we will get a glimpse of origins of replications, and specifically, we will explore them in the context of bacteria, archaebacteria, and eukaryotes and that will be mainly yeast and mammals. Let’s start by highlighting the motivation to study the origins of replication. This is actually fairly straightforward. DNA as a storage molecule is double stranded, and if you want to replicate the double stranded molecule, you will have to unwind it into
0:32
single strands. And the origin of replication as we will see, helps in this process. To get an intuition about the origin of replication, let’s actually start with a tractable case of eukaryotes, because they have a linear chromosome. And for simplicity, consider a chromatid. This is the structure that you have probably seen in textbooks. And this has two open ends, which are called telomeres… and by open I mean that they have overhangs
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that can be used directly for potential unwinding. This may in simplicity look something like this but in reality, the telomeres have a T-loop cap, which is just a folded structure. But the point is that there is a 5’ end available if you were to unzip the DNA at the telomeres. And you can do this same process on both sides. By the way, if you haven’t noticed a technical mistake, I started out by suggesting “chromatid” for simplicity.
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But chromatid is a condensed structure found in mitosis. DNA replication occurs in the interphase and there is no chromatid in the interphase. If you actually look at the DNA in the interphase, it is present as a somewhat organized hairball. In any case, the point remains that you have two ends on each chromosome that can be used to prime and start DNA replication. And this replication then has an inward direction. But if we take this intuition we run into
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the problem of different chromosomes and different timings of replication for each chromosome. Also, different chromosomes have different lengths, so the speed of replication has to be calibrated for each chromosome. You see, this gets messy, so you can’t just start from the ends - at least it does not work intuitively. Eukaryotes have linear chromosomes, but if we look at the prokaryotes, they have circular chromosomes. So there is no way to
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start replication from an available end, because there are no free ends or an overhang in a circular chromosome. This means that the replication must start internally. Which means that the chromosomes or DNA specifically, must have a way to open internally to allow the replication to proceed in some direction. And it is this internal site or a DNA sequence specifically, that is called the origin of replication. In an organism you need at least one origin of replication, but in some complex
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ones, as we will see later, you can have thousands of origins of replication. The postulation of origin of replication came from a model called replicon model, back in 1963 through studies in bacteria by sydney brenner, francois jacob and some other prominent scientists. It suggested that in the bacterial chromosome, there is a site that allows for the chromosome to be replicated and that contains the origin of replication. Now note that the bacteria
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has a single chromosome. A replicon model suggested that each bacterial chromosome has one origin or “ori” for short, and this chromosome then can also be called a single replicon because it replicates itself. So a bacterial chromosome is also a replicon. Now contrast this with eukaryotes, where the chromosomes are very large and linear. They have multiple origins for each chromosome, which can independently start replication,
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which means this part is one replicon, this other part is another replicon, and so on. So in eukaryotes, you will have one chromosome that can have multiple replicons in it. Okay, let’s get back to the replicon model. The replicon model suggested that there is a replicator sequence and an initiator factor, and both are required for the replication initiation. The replicator is a DNA sequence, whereas the initiator is a protein factor.
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Before we dive into the details and complexity of the origins, let's get a general idea about the replicator and initiator. We will start with the replicator. As we just said, replicators are DNA sequences, but they are sequences that aid in the recognition of the origin. So consider this piece of DNA, which can attract some proteins and license that region as an origin, then this piece of the DNA is called a replicator. Within the replicator, you start
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to unwind the double strands into single strands… and that particular location of unwinding is called the origin of replication, which is THE starting point for replication. So origin or ori is part of the replicator sequence. Analogically, this is similar to the situation in transcription, where you have the promoter and a TSS. The promoter is the recognition sequence for starting transcription, but the actual start point of transcription is at
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the TSS. The other part of the replicon is the initiator. This can be one or more proteins that recognize the replicator. Once again, the replicator is the piece of DNA that aids in recognition, and the recognition is done by these proteins that bind to it. So initiators have motif binding potential - they recognize the replicators. And once they do that, the initiator can recruit other proteins like helicases and polymerases and initiate replication.
6:07
A very subtle but important point is that DNA polymerase and helicases DO NOT actually recognize any specific DNA sequence motifs. In this respect, these initiators are important… since they are the only proteins to have some DNA specificity. To make this point very clear, let’s again compare this with the transcription, where you have the promoter and TSS. The transcription factors recognize the promoter sequences, and they in turn recruit RNA polymerase onto
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the promoter. The RNA polymerase by itself does not recognize the promoter. One last thing before our deep-dive into origins of replication. I want to stress that the replicon model was meant mostly for bacteria. The origins in eukaryotes as we will see later are slightly more complicated and do not really fit the constraints of a replicon model. Now, we are ready to take a deep-dive into the bacterial replicator and initiator. We
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will start with the replicator first. Bacteria have a single replicator, as we just said, they have one replicon, one origin and one chromosome. The replicator is called origin - oriC for short. This stands for chromosomal replication origin. The replicators across different bacteria can be very diverse in length. They can go from 250 base pairs to as big as 2000 bases. But in common they are all recognized by the same DnaA protein. As
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we will see shortly, the DnaA protein is the initiator protein. Back to the replicators for now… they can be either continuous or bipartite. Let’s understand the structure of a replicator by taking the example of E. coli, which has a replicator of around 250 nucleotides in length, so a fairly compact replicator. This replicator has some signature components. First, it has three sequence repeats, which are 13 base pairs in length. They are
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sometimes just called left, middle and right motifs. And together, they make up the DUE element, which stands for the DNA unwinding element. Then you have four more important motif repeats, which are 9 base pairs in length. They are simply named as R1 R2 R3 and R4, and they have a specific directionality to them. This entire region is called DAR, which stands for DnaA assembly region. These four regions are high affinity sites for DnaA protein.
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Actually they are always bound by DnaA proteins. Within this DAR region, you also have some other small sequences which are also about 5-9 nucleotides in length, that look like the R1 R2 R3 and R4 regions. The names of these internal sites are not that important but they are just named as R5, tau, I and C sites. These internal sites are low-affinity binding sites for DnaA protein. The binding of DnaA to these other sites is ATP-dependent.
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As we will shortly see, DnaA has an ATPase subunit. There are some other sites within the DAR region that regulate the activity of DnaA proteins, and these are IHF and FIS sites. We will discuss the function of IHF and FIS when we talk about the mechanism of DNA unwinding in a different video. The last part of the replicator is this newly discovered region between DAR and DUE, which is named as DnaA trios, which contains multiple GAT
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motifs. The function of this region is to aid in DnaA oligomerization into the DUE region, and this helps in the unwinding of the DUE. Since DUE is unwound, we can say that DUE is the actual origin of the replication. So in summary, the DnaA protein binds to DAR regions, and unwinding occurs at the L M R DUE regions. The DUE region after it is unwound, is also the site for single-strand protein binding. Details of all this will come later
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when we discuss the exact mechanism. For the sake of replicator, you just have to take note of the DUE, trios and DAR region. One side note. I mentioned this bipartite origin in the beginning, so let me make this clear. In other bacteria, the general structure of the replicator is very similar in a broad sense, that you have a DUE region and a DAR region. The length of these DARs and DUEs can be variable but the syntax is pretty much
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the same. In some cases, for instance, the helicobacter pylori, the origin is split into two parts, so it is a bipartite structure. One part of the origin is a DAR region exclusively, and then at some distance, there is another part which contains another DAR and the DUE region. So it has this split structure of origin, and that is why it is called bipartite structure. Alright, that was the replicator… Now we get to the initiator of the replicon. As we
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said already, the DnaA protein is the initiator in bacteria. It is common to all known bacteria. The structure of DnaA is fairly simple. It has four domains. One is an oligomerization domain. Two is a flexible linker. Three is the ATPase domain, which is of the AAA+ family. The triple A stands for ATPase associated with various cellular activities. And this is where the AAA comes from. The plus symbol is a nomenclature to differentiate it from
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the non-plus AAA family. This domain also binds the single stranded DNA… and DUE gets unwound, so that's one binding target for this particular domain. The third domain also recognizes in some capacity the trios region. The main recognition of the DnaA however, of the replicator comes from the fourth domain, which contains a helix turn helix region. This one is very important… it functions to bind the double stranded region… more
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specifically, it binds to the DAR region 1, 2 3 and 4. And this snapshot completes our discussion of the bacterial origin of replication. Let’s now talk about archaebacteria. They typically have one to four replicators per chromosome. Their recognition sequences are called Origin recognition boxes or ORBs for short, and they are very diverse in composition. As an example, if this is an archaea chromosome, then it can have anywhere from one to four origins, that may or may not be different
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from one another. If we zoom into one of these origins, you will find a DUE element, and a bunch of recognition motifs called ORBs. And as I said, these ORBs can be different from each other - that is what the colors represent here. So that’s the general structure of the replicator. The initiator in archaea is called orc1 or cdc6. This protein binds ORBs. In archaebacteria, there can be multiple paralogs of orc1, conveniently named as 1
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2 3 and so on. These paralogs are different in their affinities to different ORBs. Orc1 looks quite similar to a DnaA protein, in that it has a AAA+ domain and a Winged helix domain, and this serves as the DNA binding protein. Now let's put this in context to the replicators. The different Origin recognition complexes in one or more combinations can recognize specific ORBs. So a replicator can have multiple paralogs bound to it. This together
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unwinds the region, which is the DUE region, and that can recruit helicases, polymerases and then start the DNA replication. Alright, that is the context of archaebacteria. And now let’s move onto the eukaryotic replication origin. Eukaryotes have many many origins. Broadly speaking, eukaryotic replicators or origins can be split into yeast and everything non-yeast. Let’s go over yeast first. They have a 12 Mb long genome, and when I say yeast
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it encompasses all the known Saccharomycotina species like cerevisiae, pombe, and others. Yeast is the only known eukaryote with a very specific consensus DNA sequence for origin of replication. And the replicator in the yeast is called ARS, which is short of autonomously replicating sequence. There are about 300-400 of these in the entire yeast genome. Let’s sketch it out. It has four important motifs - the A motif, which is about 11 bases long,
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and the other motifs are variants of the B motif, named B1, B2 and B3. The A and B1 is the core sequence, which recruits ORC. The A motif has a conserved sequence, which is fairly AT rich. This entire region is around 100-200 bases long. And because now we are in eukaryotes, you have to note that the DNA is maintained as a chromatin, so you have histones that may be important to the origin recognition. Functionally speaking, the core
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A and B1 motifs recruit the initiator ORC, the B2 motif potentially recruits helicase. The function of B3 is somewhat unclear except from the fact that it recruits this Abf1 factor, but it doesn’t seem to be essential. So it is not clear why or how this is important. The initiator in case of yeast is this ORC, which stands for origin recognition complex. It seems to be conserved in all eukaryotes. It is a complex of 6 proteins, which together
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form a ring shaped complex. Just to intuitively give you an idea, I will sketch this out. The ARS recruits the ORC at the A and B1 motif. Specifically, the Winged helix domain recognizes the core. The ORC also has AAA+ domains. So most of the binding is done at these A and B1 motifs. The ORC can also interact with some specific histone marks, but they may not be functionally as important. Once this recognition is complete, the origin is opened,
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helicase is loaded and replication can start. So that was yeast, and now let’s consider the rest of eukaryotes, and I will limit the scope and generalize them by considering mammals. Outside of yeast, the eukaryotic origin of replication is actually quite puzzling. It is still an active area of research because there is no-clear consensus on the replicator sequence - people haven’t found it or maybe it doesn’t exist. But it is clear that origins
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are present at some specific locations in the genome. Usually they are in the order of thousands, but only 10-20% of them are actually used. This phenomenon is called origin licensing. The origin definition in eukaryotes depends on multiple factors. The lack of consensus is thought to be overcome by some other variables such as DNA accessibility, AT or GC rich regions, CpG islands, nucleosome positioning, epigenetic marks, and even DNA topology, which is typically
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G4 motifs, also known as G quadruplexes. These multiple factors make-up the prevalent view of the origin of replication in eukaryotes. Let’s see what this means… places of origin of replication means that there are regions which in some capacity contain these elements and “somehow” recruit ORCs at these locations. The ORC being the initiator protein complex. These ORCs can interact with histone marks such as H4K20 methylation. Elements nearby
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can also recruit some other specific proteins, and the synergy of these elements leads to helicase recruitment, and then opening of DNA can happen and initiation of replication can occur. And this region where all these occur is called the initiation zone, which is around 30-40 kilobases in length. There is another view that has recently gained precedence, which mainly relies on 3D genome topology, and specifically, the topologically associating
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domains which are in context. These dynamic regions are DNA, which are much more frequently interacting with each other. The edges or borders of TADs are marked by CTCF proteins, and the dynamic organization is mediated by a motor protein called cohesin. The point here is that the borders, which are often 40-50 kb in length, are highly enriched for CTCF protein binding. The movement of cohesin and concentration of CTCF in these locations,
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is thought to create a permissive environment for helicase or ORC loading at these borders. The mechanism at play is called loop-extrusion, which is the name for the movement done by cohesin. How exactly the loading happens is unclear. This may all sound wishy washy and directionless… and if it does, that is exactly the point. As I said in the beginning, this is an active area of research. Anyways, this completes our discussion on the origin of
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