BIOCHEMISTRY DISCUSSION 8

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So now we are going to take a closer look at DNA replication.

Created by Brett Barbaro

Biochemistry: A Short Course

Fourth Edition

CHAPTER 34 DNA Replication

Tymoczko • Berg • Gatto • Stryer

DNA is replicated by protein complexes called polymerases. And the replication of DNA is a very complex process and involves a number of moving parts.

Chapter 34: Outline

34.1 DNA Is Replicated by Polymerases 34.2 DNA Replication Is Highly Coordinated

Let’s start off by watching an animation of replication. This excellent animation will give you an overall idea of what’s happening and then we can go into the individual steps one by one in more detail. (https://www.dnalc.org/resources/3d/04- mechanism-of-replication-advanced.html)

https://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html

We’re going to talk about DNA replication in E. coli, because it’s much more simple than it is in human beings and this will give you some basic ideas of how the process works. Even in E. coli it’s very complicated, with over 20 proteins - and 5 of these proteins are called DNA polymerases which actually do the catalysis of adding new bases to a growing daughter strand of DNA.

Section 34.1 DNA Is Replicated by Polymerases

Learning objective 3: Identify the enzymes that take part in the process of DNA replication.

• DNA replication in E. coli requires more than 20 proteins, 5 of which, called DNA polymerases, catalyze the synthesis of new DNA.

These 5 different polymerases each have slightly different functions. Polymerase I is responsible for removing primers and repairing DNA, and Polymerase III is responsible for the majority of the replication. Polymerases II, IV, and V are specially designed for repairing DNA after it’s been damaged.

Table 34.1 E. coli DNA Polymerases

Very simply, DNA polymerization involves the addition of a new NTP molecule (that’s ATP, CTP, GTP or TTP - it’s a triphosphate nucleotide version of the nucleoside bases), and that is done by breaking the bond between the alpha and beta phosphates and integrating the alpha phosphate and its attached ribose and base to the growing chain. There are some basic characteristics of DNA synthesis. You need to have NTPs in order to do it - to match the complementary sequence and to build your new strand out of. And magnesium plays a big role in this process. Actually NTPs are often associated with magnesium. The three negatively charged phosphates tend to associate readily with positively charged magnesium ions. A template strand is needed, which means you have to have an original strand of DNA to build a second strand upon. And you need to have a primer or an existing stretch of polymerized nucleic acid in order to continue the production. In addition to that, many of these DNA polymerases can remove mismatched bases and correct mistakes that have been made.

DNA Polymerase Catalyzes Phosphodiester-Linkage Formation (1/2)

• The reaction catalyzed by DNA polymerase is

• Key characteristics of DNA synthesis are 1. Four deoxynucleoside triphosphates

and Mg2+ are required. 2. A template strand is used to direct

DNA synthesis. 3. A primer from which the new strand

grows must be present. 4. Many DNA polymerases have

nuclease activity that allows for the removal of mismatched bases.

DID YOU KNOW? A template is a sequence of nucleic acids that determines the sequence of a complementary nucleic acid.

DID YOU KNOW? A primer is the initial segment of a polymer that is to be extended on which elongation depends.

Figure 34.1 A polymerization reaction catalyzed by DNA polymerases.

This diagram will show you very simply how the process works. You have an existing polymer on the top - you see, on the left hand side, we have the template strand on the bottom and the primer on the top. And a dATP (deoxy-ATP) molecule arrives, and is attached, and two phosphates (inorganic pyrophosphate) is released. And then to match the next base (the cytosine) we have a dGTP coming in, and that gets added with release of another pyrophosphate.

Diagram of a Polymerization Reaction Catalyzed by DNA Polymerases

Figure 34.2 Strand-elongation reaction. DNA polymerases catalyze the formation of a phosphodiester bridge. Elongation of the DNA strand proceeds in the 5¢-to-3¢ direction.

The reaction is actually quite simple. It takes the 3’ oxygen and that oxygen attacks the alpha phosphate group on the NTP. And that attacking releases the two other phosphates and is a highly exergonic reaction so it’s very likely to take place.

Diagram of the Strand-Elongation Reaction

It still is helped along though by the existence of these polymerases, and the active site of a polymerase is like the palm of your right hand.

DNA Polymerase Catalyzes Phosphodiester-Linkage Formation (2/2)

• The structures of DNA polymerases are similar in that the region containing the active site approximates the shape of a right hand.

• The thumb and finger domains wrap around the DNA, positioning it in the active site located in the palm domain.

From; https://image.slidesharecdn.com/dna-140307101952-phpapp01/95/dna-46-638.jpg?cb=1394187701

Figure 34.3 DNA polymerase structure. The first DNA polymerase structure determined was that of a fragment of E. coli DNA polymerase I called the Klenow fragment. Notice that, like other DNA polymerases, the polymerase unit resembles a right hand with fingers (blue), palm (yellow), and thumb (red). The Klenow fragment also includes an exonuclease domain that removes incorrect nucleotide bases. [Drawn from 1DPI.pdb.]

The existing template strand goes along in through that “palm” section and the “thumb and fingers” sections wrap around the template strand and push together the new incoming nucleotide and facilitate the reaction.

Diagram of the DNA Polymerase Structure

So, obviously, in order to create a complementary strand you need to have complementary bases. And those complementary bases are recognized by the DNA polymerase because they fit into the active site along with the existing template. The template is matched not only by the hydrogen bonding but also by just the overall shape. And once the proper shape is in there, the hand - the thumb and finger domains - can fold over and create a pocket for the reaction to take place.

The Specificity of Replication Is Dictated by the Complementarity of Bases

• The specificity of replication is determined not only by the correct hydrogen bonding between the incoming dNTP and the DNA template but also by the overall shape of the incoming base.

• Binding of the incoming dNTP induces a structural change in which the finger domain closes over the nucleotide, forming a pocket into which only the correct base pair fits.

Figure 34.4 Shape complementarity. The base analog on the right has the same shape as adenosine, but groups that form hydrogen bonds between base pairs have been replaced by groups (shown in red) not capable of hydrogen bonding. Nonetheless, studies reveal that when incorporated into the template strand, this analog directs the insertion of thymidine in DNA replication.

This is just an example of the idea that it’s not always the hydrogen bonding that matters. You have here on the left the regular adenosine base, and on the right the areas in red have been changed so that they are of similar shape but they no longer are able to hydrogen bond. This altered adenosine, though, will be able to direct the insertion of a thymidine during DNA replication because thymidine will still fit. One might guess, though, that this reaction would not be as energetically favorable, because of the lack of hydrogen bonding, and therefore a lot less likely to take place.

Diagram of Shape Complementarity

Figure 34.5 Shape selectivity. The binding of a deoxyribonucleoside triphosphate (dNTP) to DNA polymerase induces a conformational change, generating a tight pocket for the base pair consisting of the dNTP and its partner on the template strand. Such a conformational change is possible only when the dNTP corresponds to the Watson–Crick partner of the template base. Only the part of the polymerase directly participating in nucleotide binding is displayed (yellow ribbons). The green ball represents the Mg2+ ion. [Drawn from 2BDP.pdb and 1T7P.pdb.]

Here is an illustration of the active site, and you can see (in red) the template strand, and (in blue) the growing daughter strand, and the pocket that is formed where the template strand and the 3’ end of the growing daughter strand come together in the active site of the protein polymerase. And after a complementary nucleotide drifts into that space, the pocket is able to close around it. In this diagram you can also see the green circles which represent magnesium ions.

Diagram of Shape Selectivity

Figure 34.6 Werner syndrome. A very rare disorder, Werner syndrome is characterized by premature aging. (A) People afflicted by Werner syndrome develop normally until adolescence, at which time they begin to age rapidly. (B) By age 40, they look several decades older. [Courtesy of the International Registry of Werner Syndrome, www.wernersyndrome.org.]

Now there are three basic things that need to take place in order for DNA to be replicated. And the first thing is for the two strands to be separated. The separation of the two strands is done by enzymes called helicases. And this is powered by ATP hydrolysis, so it actually uses up quite a bit of energy. The helicase (and I think we are talking still specifically about an E. coli helicase) is a ring-like structure of 6 subunits.

Clinical Insight: The Separation of DNA Strands Requires Specific Helicases and ATP Hydrolysis

CLINICAL INSIGHT The Separation of DNA Strands Requires Specific Helicases and ATP Hydrolysis

• Helicases, powered by ATP hydrolysis, separate the strands of the double helix to make the DNA available for DNA polymerase.

• The helicase, which consists of a ringlike structure composed of six subunits, acts as a wedge to pry the helix apart.

• Defects in the helicase can result in Werner syndrome, a pathological condition characterized by premature aging.

Figure 34.7 Helicase mechanism. One of the strands of the double helix passes through the hole in the center of the helicase, bound to the loops of two adjacent subunits. Two of the subunits do not contain bound nucleotides. On the binding of ATP to these two subunits and the release of ADP + Pi from two other subunits, the helicase hexamer undergoes a conformational change, pulling the DNA through the helicase. The helicase acts as a wedge to force separation of the two strands of DNA.

One strand of the DNA, the mother DNA, goes through the middle of the helicase and gets passed along from subunit to subunit along with the hydrolysis of ATP. As it gets passed along it proceeds down the strand of DNA and separates it from the other strand.

Diagram of the Helicase Mechanism

This unwinding can cause some problems in the DNA though, because it introduces supercoils. Topoisomerases are enzymes that are specifically there to induce or eliminate supercoils. So what they’ll do is they will nick one of the strands of DNA, and actually rotate the remaining strand around until the first nicked strand is back together, and then reseal it.

Topoisomerases Prepare the Double Helix for Unwinding

• When DNA is unwound for replication, supercoiling occurs because of overwinding in nearby regions of the helix.

• Topoisomerases are enzymes that induce or eliminate supercoils.

• Type I topoisomerases relax supercoils, whereas type II topoisomerases such as DNA gyrase in E. coli introduce supercoiling.

DID YOU KNOW? To gyrate is to move in a circle or spiral or to revolve, usually about a fixed point or on an axis.

Figure 34.8 Consequences of strand separation. DNA must be locally unwound to expose single-stranded templates for replication. This unwinding puts a strain on the molecule by causing the overwinding of nearby regions.

As the helicases proceed from the left to the right, they unwind the strands of DNA - but that introduces tension and twisting in the DNA ahead of it, which needs to be relieved by the action of topoisomerases.

Diagram of the Consequences of Strand Separation

Another domain in many polymerases actually can remove nucleotides from a growing strand. If the two strands get separated for some reason, it might be because of some sort of a mismatch - there might have been some sort of incorrectly inserted nucleotide. Well that’s not good! And if the strands are not together, then that nucleotide is able to drift apart and flop around, and eventually (probably) reach the exonuclease site on the polymerase.

Many Polymerases Proofread the Newly Added Bases and Excise Errors

• Many polymerases also have nuclease activity that is used to remove mismatched nucleotides.

• The E. coli enzyme DNA polymerase I, which is used in replication and DNA repair, has exonuclease activity that removes mismatched nucleotides from the 3’ end of the DNA.

• An incorrectly inserted nucleotide moves from the polymerase active site to the nearby exonuclease active site, where it is hydrolytically cleaved.

Figure 34.10 Proofreading. The growing polynucleotide strand occasionally leaves the polymerase site, especially if there is improper base-pairing, and migrates to the active site of exonuclease. There, one or more nucleotides are excised from the newly synthesized strand, removing potentially incorrect bases.

This is just another example. If you have a mismatched base there on the blue growing strand, it won’t stick to the other strand and will flop over into the exonuclease site and be removed.

Diagram of Proofreading

So if I were to ask you what are the three key enzymes required for DNA synthesis, and what biochemical challenges in replication do they address, what would you say? I suggest you pause the recording for a second and think about it. Well the answer would be: you need helicase, topoisomerase, and polymerase. The helicase is there to separate the strands, the topoisomerase is there to relieve the tension that is created by the separation of the strands, and the polymerase is doing the actual work of strand elongation by sequentially adding new nucleotides to the daughter strand.

Quick Quiz 1

QUICK QUIZ 1 What are the three key enzymes required for DNA synthesis, and what biochemical challenges to replication do they address?

Now, DNA replication is actually enormously fast. The E. coli genome can be replicated in less than 40 minutes - so that means that it goes about 2,000 nucleotides per second. Now, this is pretty typical of molecular reactions - they are very fast! Things are banging around into each other at alarming speeds. But DNA polymerase has a special advantage in that it is physically attached to the DNA strand, and so they are not able to drift away, and so they just keep interacting over and over again. We’ll go into more detail on that in a second.

Section 34.2 DNA Replication Is Highly Coordinated

Learning objective 4: Describe how replication is organized and distinguish between eukaryotic replication and bacterial replication.

• DNA replication is rapid.

• The E. coli genome, consisting of 4.6 million base pairs, is replicated in less than 40 minutes, a rate of 2000 nucleotides per second.

Now, where does this replication start? And if you recall E. coli, which is the example that we are using right now, has a circular genome - so it needs to pick some place on that circle to go. And it turns out that there is a special location {the “origin of replication”} called the oriC locus, which has 245 base pairs, and is recognized by these DNA replication enzymes which then assemble on the DNA and begin to replicate. The first one that binds to this site is called DnaA, and it’s basically just a locator that finds the locus - and then DnaB, which is a helicase, actually attaches to that DnaA and begins to unwind {the DNA}. Single stranded binding proteins (SSB) attach to AT-rich sites in this locus to prevent the two strands from coming back together. Single stranded binding proteins are also important throughout the process of replication. At this point it is called a “prepriming complex” because the priming has not actually begun.

DNA Replication in E. coli Begins at a Unique Site

• Replication begins in E. coli at a unique 245–base pair region called the oriC locus.

• DnaA protein binds at distinct sites in the locus and then recruits DnaB, a helicase that uses ATP hydrolysis to unwind the locus.

• Single-stranded-binding proteins (SSB) bind to the AT-rich sites in the locus to prevent reannealing of the helix.

• The resulting complex of DNA and proteins is called the prepriming complex.

Figure 34.11 The origin of replication in E. coli and formation of the prepriming complex. (A) The oriC locus has a length of 245 bp. It contains a tandem array of three nearly identical AT-rich regions (green) and four binding sites (orange) for the DnaA protein.

So this is a linear representation of the oriC locus. And this has 245 base pairs, and there are 4 separate binding sites for DnaA protein. And then, on the left, you see the green AT-rich sequences that are each 13 nucleotides long, and that’s where the DNA actually starts to be pulled apart. It’s worth noting that the connections between the A and T are actually weaker than the connections between G and C, because there are only two hydrogen bonds between adenine and thymine where there are three hydrogen bonds between guanine and cytosine. So that makes areas that are AT rich in DNA easier to pull apart.

Diagram of the Origin of Replication in E. coli and Formation of the Prepriming Complex (1/2)

Figure 34.11 The origin of replication in E. coli and formation of the prepriming complex. (B) Monomers of DnaA bind to their binding sites in oriC and oligomerize, wrapping the DNA around the oligomer. This structure marks the origin of replication and favors DNA strand separation in the AT-rich sites (green). (C) The AT-rich regions are unwound by DnaB and trapped by the single-stranded-binding protein (SSB). At this stage, the complex is ready for the synthesis of the RNA primers and assembly of the DNA polymerase III holoenzyme.

So DnaA monomers will bind to these binding sites in the oriC, and then associate with each other to create a complex structure that can be recognized by other proteins to assemble the rest of the DNA replication apparatus. Why does it do it in this particular way? Well, this is actually just a hypothetical drawing. I think that it’s not really known how that works. And this is also supposed to just be E. coli. So I wouldn’t read too much into this flower shape. The main point is that it creates some sort of structure, which is then recognized, and the DnaB complexes can then assemble and separate the strands of DNA, as you can see on the right, and the single stranded binding proteins (or SSBs) then attach to the single strands of DNA to make sure that they don’t come back together.

Diagram of the Origin of Replication in E. coli and Formation of the Prepriming Complex (2/2)

Now the first thing that happens next is actually the synthesis of an RNA primer. I think it may be because RNA is easier to synthesize than DNA - I am not actually sure exactly why it has to be this way, but this is what happens. This RNA polymerase will come in - it’s called primase - and it synthesizes a very short RNA sequence, and then falls off. So now you’ve got an RNA sequence. That serves as the primer for the DNA synthesis. This priming is actually a pretty involved process - there’s a lot of proteins involved, so much so that they have named this complex the “primosome”. And you can imagine that such a large complex will be subject to a great deal of regulation. Everything needs to be in the right place at the right time in order for priming to begin. And that makes a certain amount of sense - because once you’ve started priming, then you’re ready to start replicating the entire genome. And once you’ve started replicating then there’s really no stopping - and it’s quite a long, involved, and energy consuming process. So you want to make sure that everything is in the right place. And that’s what the primase does, is it coordinates all of the different signals from around the cell and only primes the DNA when it’s ready to replicate. After you’ve replicated the entire DNA, or even before that perhaps, you’ve still got this short stretch of RNA in there, and that gets removed by DNA polymerase I, which is a 5’ to 3’ exonuclease - which just means it starts at the 5’ end of the RNA and removes bases until it gets to the end of the RNA, replacing them with DNA bases.

An RNA Primer Synthesized by Primase Enables DNA Synthesis to Begin

• A special type of RNA polymerase called primase synthesizes a short stretch of RNA (≈10 nucleotides) complementary to a DNA strand that then serves as a primer for DNA synthesis.

• The primase is part of a large complex called the primosome that joins the prepriming complex to synthesize the required primer.

• DNA polymerase I, employing a third enzymatic activity, a 5’ à 3’ exonuclease activity, subsequently removes the primers and replaces the RNA with DNA.

Figure 34.12 Priming. DNA replication is primed by a short stretch of RNA that is synthesized by primase, an RNA polymerase. The RNA primer is removed at a later stage of replication.

Here’s just a little diagram showing how that works. You start off with a DNA template (and in this case we are talking about a single strand of DNA). Primase will come in and make an RNA primer, and then from that the DNA polymerase will make new synthesized DNA. Now kind of an interesting point here - the DNA template as you can see on the left hand side is the 3’ end and the right hand side is the 5’ end. So the RNA and DNA are synthesized from the 5’ end to the 3’ end, but that means that they actually are traveling along the template from the 3’ end to the 5’ end.

Diagram of Priming

The place where the DNA is being split apart and replicated is called the replication fork. And it moves along the DNA in one direction and it copies both strands simultaneously. But as we just mentioned DNA is only synthesized in the 5’ to 3’ direction - and if you’ve got two strands, you’ll be traveling along one strand in the 5’ to 3’ direction, and you’ll be traveling along the other strand in the 3’ to 5’ direction. So one of the strands is synthesized (replicated) continuously, and that’s the one that you’re actually traveling along in the 3’ to 5’ direction. The other strand has to be made in individual segments called Okazaki fragments, and you saw this example in the video.

One Strand of DNA Is Made Continuously and the Other Strand Is Synthesized in Fragments • The site of replication is called the replication fork. • The fork moves in one direction, demonstrating that both

strands are copied simultaneously. • However, all DNA polymerases synthesize DNA only in the 5’

à 3’ direction. • At the replication fork, one strand is synthesized

continuously in the 5’ à 3’ direction. • The other strand is synthesized discontinuously as small

pieces, called Okazaki fragments, in the 5’ à 3’ direction.

• The discontinuous assembly allows fork movement in the 3’ à 5’ direction while fragments are made in the 5’ à 3’ direction.

Figure 34.14 Okazaki fragments. At a replication fork, both strands are synthesized in the 5¢ → 3¢ direction. The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short pieces termed Okazaki fragments.

Well, here’s a diagram of that. The replication fork is right there in the middle, the parental DNA extending to the left. And the leading strand is synthesized continuously in one long stretch, whereas the lagging strand is synthesized in small bursts called Okazaki fragments.

Diagram of Okazaki Fragments

Most of the DNA synthesis taking place in E. coli is done by DNA polymerase III, and once it starts it attaches to the DNA and doesn’t let go. Well, the way that it attaches is using what’s called a “sliding clamp”. It’s a dimer of proteins that wrap around the DNA. And this clamp gets loaded onto the DNA by another protein called the “clamp loader”, at the expense of some ATP hydrolysis. Once again, you know - DNA replication takes a lot of energy.

DNA Replication Requires Highly Processive Polymerases

• The enzyme responsible for the majority of DNA synthesis in E. coli is DNA polymerase III.

• DNA polymerase III is highly processive, meaning that once it begins catalysis, it rarely releases the DNA substrate.

• The source of the processivity is the sliding clamp (β2 subunit), a dimer that encircles the helix.

• The sliding clamp is loaded onto the DNA by a protein called the clamp loader, which uses the energy of ATP hydrolysis to open and close the clamp.

Figure 34.15 The structure of a sliding DNA clamp. The dimeric b2 subunit of DNA polymerase III forms a ring that surrounds the DNA duplex. Notice the central cavity through which the DNA template slides. Clasping the DNA molecule in the ring, the polymerase enzyme is able to move without falling off the DNA substrate. [Drawn from 2POL.pdb.]

So this an illustration of the clamp (the sliding clamp). There are two subunits - the yellow one on the left, and the orangish one on the right, and the DNA slides through the center.

Diagram of the Structure of a Sliding DNA Clamp

As mentioned before, the leading strand of DNA is synthesized continuously, and the lagging strand is synthesized discontinuously as Okazaki fragments. The lagging strand is looped around so that it passes through the active site in the proper orientation. So the DNA gets pulled apart and the lagging strand begins to loop out. It is still attached at both ends to the replication fork, but a large portion of it is being extruded. And when the Okazaki fragment is finished being made, then that loop gets released, and a new loop begins to form, and a new Okazaki fragment begins to be created. Each one of these Okazaki fragments (in E. coli) is about 1,000 nucleotides in length. And this loop coming out and going back in continuously looks a little bit like a trombone, so they call it the “trombone model”. Now once an Okazaki fragment has been finished, and the polymerase reaches the previously synthesized daughter strand, it still needs to be joined to that previously synthesized daughter strand. And that’s not something that polymerase does, that’s done by a different enzyme called DNA ligase (which...that’s its basic job, is just to go around the cell and repair any nicks in DNA - so if there’s any damage done to the DNA, then the ligase will come and it will fix it) and that’s what creates the final molecule of DNA out of the Okazaki fragments.

The Leading and Lagging Strands Are Synthesized in a Coordinated Fashion

• The strand of DNA that is synthesized continuously is called the leading strand.

• The lagging strand is synthesized discontinuously as Okazaki fragments.

• The lagging strand is looped so that it passes through the polymerase active site in the 3’ à 5’ direction, allowing synthesis to occur in the 5 ’à 3’ direction.

• After DNA of ≈1000 nucleotides in length is synthesized, the loop is released and a new loop is formed, a process called the trombone model.

• DNA polymerase I removes the RNA primer and replaces the sequence with DNA.

• DNA ligase joins the fragments to yield an intact strand.

Figure 34.17 The DNA polymerase holoenzyme. Each holoenzyme consists of two copies of the polymerase core enzyme linked to a central structure. The central structure includes the clamp-loader complex, which binds to the hexameric helicase DnaB.

So this would be a cartoon example of the DNA polymerase complex. And you have the helicase up at the top, the clamp loader there in green, and the polymerase core - two of them are associated with this. And those yellow rings that you see attached to the polymerase cores would be the clamps that hold the polymerase to the DNA.

Diagram of the DNA Polymerase Holoenzyme

Figure 34.18 The trombone model. The replication of the leading and lagging strands is coordinated by the looping out of the lagging strand to form a structure that acts somewhat as a trombone slide does, growing as the replication fork moves forward. When the polymerase on the lagging strand reaches a region that has been replicated, the sliding clamp is released and a new loop is formed.

Here is a diagram of it all going together. And you need that clamp loader in there because the lagging strand that’s making the Okazaki fragments is being continually removed and reattached, and each time that happens you need to reattach the clamp.

Diagram of the Trombone Model

So now look another time at that illustration from David Goodsell of the DNA inside of an E. coli. And you can see near the center of the illustration a little down and to the right - that would be the polymerase complex. And you can see the double- stranded DNA, coming in from the bottom of the complex, being separated into two strands. One of the strands is being synthesized continuously and it looks all double stranded, while the other strand is wrapped around a bunch of single-stranded binding proteins {SSBs} that are keeping it single stranded. And the Okazaki fragment is there to the left of those SSBs coming out of the DNA polymerase complex, in this diagram, in the same direction. Well, out of the DNA polymerase complex on the bottom is another single-stranded section that is bound to SSBs. Those will be stripped away and the daughter strand will be synthesized on that template. It will end at the place where it is again double stranded, where the daughter strand has already been synthesized, where it will drop off. And then the polymerase complex will reattach to the single stranded DNA up at the top, and the ligase will come and connect the Okazaki fragment to the growing daughter strand.

Figure 34.19 The DNA ligase reaction. DNA ligase catalyzes the joining of one DNA strand with a free 3¢-hydroxyl group to another with a free 5¢-phosphate group. In eukaryotes and archaea, ATP is cleaved to AMP and PPi to drive this reaction.

This is just an illustration of the process of DNA ligase. And remember - when you put on these nucleotides {during synthesis} you are basically hydrolyzing {the nucleotide}. So it takes a little bit of energy to do this. So DNA ligase requires ATP in order to make that bond.

Diagram of the DNA Ligase Reaction

Now, eukaryotic DNA is much more complicated. We saw in the packaging it’s much more complicated – well, also in its synthesis. One of the situations that we have is that it is really, really long - a lot longer. And another one is that it’s not circular, either. So, to deal with the problem of it being so much longer, you have more than one origin of replication - in fact you have about 30,000 in humans. And so they will be replicated sometimes simultaneously, sometimes at different times. And how does the replication machinery know if its already been replicated or not? Well, it uses these proteins called “licensing factors” that will attach to the replication origins after replication has taken place and that will prevent them from taking place again. DNA polymerase alpha, which is in eukaryotes, initiates DNA replication by creating a DNA molecule that’s only 20 nucleotides in length. {DNA pol alpha} will drop off then, and DNA polymerase delta, which is much more analogous to the DNA polymerase III in E. coli, will continue and do the rest of the DNA synthesis. So these are just a few of the differences between the prokaryotic and the eukaryotic DNA synthesis. But you can see that since the eukaryotic is a bit more complicated it was probably a good idea to start with the prokaryotic because it’s simpler.

DNA Synthesis Is More Complex in Eukaryotes Than in Bacteria

• Eukaryotes have much more DNA than bacteria does. Moreover, the DNA is present as linear molecules in multiple chromosomes.

• Multiple origins of replication are required with each origin of replication representing a replication unit or replicon. Humans have ≈ 30,000 replicons.

• Proteins called licensing factors allow only one replication per replicon per round of DNA synthesis.

• DNA polymerase α, which displays primase activity, initiates DNA replication in eukaryotes, generating a DNA molecule ≈ 20 dNTP in length.

• DNA polymerase δ, a more processive enzyme, extends the chains. The switch from polymerase α to δ is called polymerase switching.

Now, the fact that eukaryotic DNA is linear presents a couple of problems. For one thing, at the ends of the DNA, the DNA is susceptible to damage by nucleases, which are just proteins that are there to chew up loose DNA (which is important - I mean, you can’t have a bunch of loose DNA floating around, you know, it might be viral DNA). But you don’t want to chew up your own DNA. And another thing is that, because of the way that DNA synthesis works, one strand will get shorter each time the DNA is synthesized. So the DNA deals with this by having regions at the ends of the chromosomes called telomeres.

Telomeres Are Unique Structures at the Ends of Linear Chromosomes

• The free ends of linear DNA molecules present two biochemical difficulties. 1.They are susceptible to damage by nucleases. 2.Due to the nature of DNA synthesis, one strand will

shorten upon each round of DNA synthesis. • The ends of the chromosomes are called telomeres. The

longer of the two strands, which represents the leading strand, is rich in guanine. The stretch of guanine-rich single- stranded DNA can form a loop structure to protect the end of the chromosome.

Figure 34.20 Telomere shortening. Over many cycles of replication, the DNA at the telomeres would continuously shorten. [(A) After A. J. F. Griffiths, S. R. Wessler, R. C. Lewontin, and S. B. Carroll, Introduction to Genetic Analysis, 9th ed. (W. H. Freeman and Company, 2008), p. 288.

So at the top you can see the origin of replication and the Okazaki fragments being formed on the lagging strand. Now, when the replication fork reaches the end of the DNA, there are still Okazaki fragments that have not been connected yet. So the primers get degraded and there are gaps left which then need to be filled. And those gaps are easy to fill because they already have the existing Okazaki fragment to build off of --- except at the very end. And you can see that 5’ red segment there doesn’t have anything to build on. DNA can only be assembled in the 5’ to 3’ direction, so you end up with a 3’ overhang which is part of the telomeric sequence and that will continue to happen every time that you replicate.

Diagram of Telomere Shortening

Figure 34.21 A proposed model for telomeres. A single-stranded segment of the G- rich strand extends from the end of the telomere. In one model for telomeres, this single-stranded region invades the duplex to form a large duplex loop.

The telomeric sequences at the ends of eukaryotic DNA resolve these problems. Telomeres consist of a number of repeating DNA sequences, and these repeating elements are guanine rich, and they’re able to form a loop structure which can then protect the end of the chromosome from nuclease attack.

Diagram of a Proposed Model for Telomeres

Another feature of these telomeres is that they contain a specific sequence that reacts with an enzyme called telomerase. And telomerase has an RNA template in it that it can use to extend the leading strand and extend the length of the telomere. So when cells are dividing rapidly this telomerase activity is very important - because the telomeres are only so big, and if you start shortening every time you divide eventually you are going to start reaching genes that perform important functions and cells won’t be able to survive anymore. Now what sort of cells are replicating all the time? Well, cancer cells is one of them. And cancer cells are known to be “immortal”, they call them, because they are able to continue dividing and continue to be alive for many years. In fact, some of the cells that are used in laboratories called HeLa cells were cancer cells from a woman named Henrietta Lacks {THERE’S AN INTERESTING STORY HERE} and they have been used continuously for over 50 years now. So there’s some evidence that for most of your normal cells in your body, the telomeres do get shorter as you get older. And one theory of aging is that the shortening of the telomeres is what’s actually causing the slow degradation of your chromosomes and your eventual death. So a lot of interest has been generated in telomerase as a potential cure for old age or old age related illnesses. Telomerase is also extremely important when a new life is being created - after an egg and a sperm combine, telomerase creates long telomeres from the original chromosomes and then those telomeres get replicated from that point on.

Clinical Insight: Telomeres Are Replicated by Telomerase, a Specialized Polymerase

That Carries Its Own RNA Template CLINICAL INSIGHT Telomeres Are Replicated by Telomerase, a Specialized Polymerase That Carries Its Own RNA Template

• The G-rich leading strand of the telomere can be maintained by the enzyme telomerase.

• Telomerase contains an RNA template that it uses to extend the leading strand.

• In rapidly dividing cells, including cancer cells, telomeres must be maintained by the telomerase to prevent shortening of the lagging strand, which would lead to cell death.

• High telomerase activity is a characteristic of cancer cells.

Figure 34.22 Telomere formation. The mechanism of synthesis of the G-rich strand of telomeric DNA. The RNA template of telomerase is shown in blue, and the nucleotides added to the G-rich strand of the primer are shown in red. [After E. H. Blackburn, Nature 350:569–573, 1991.]

And this is just a diagram showing how the telomerase will attach to a telomere and facilitate the extension of the strand at the end using an RNA template that is part of the telomerase.

Diagram of Telomere Formation