Molecular Biology DNA Replication

Hello, and welcome back to www.educator.com, today's lecture is going to be on DNA replication.0000

As an overview, we are going to talk about the eukaryote cell cycle, once again.0008

We are going to focus on S phase because that is where DNA synthesis occurs.0012

We will talk about our major players, that will be how we recognize the origins.0018

We will talk about how strands separation occurs,0026

removing supercoils, as well as why we need to remove supercoils.0033

We will then go over the big part of the actual DNA synthesis.0037

Finally, we will go to the final processing and then we will talk about telomeres and the end replication problem.0041

The eukaryotic cell cycle, once again.0052

G1, remember that is just our growth phase.0058

That is going to be about 9 to 11 hours long.0062

S phase which is what we are going to focus on in this unit, that is where DNA replication occurs,0069

and that usually takes about 8 to 12 hours.0083

G2 phase, another growth phase is about 2 to 4 hours.0087

Mitosis is about 1 hour.0095

The normal human cell will divide on average about every 24 hours.0102

G1 is going to be the most variable timing of that stage.0112

Today, as I said, we are talking about DNA replication.0118

We are going to focus on S phase.0120

We cannot talk about DNA replication without talking about Watson and Crick.0126

Remember, back from the second unit that we talked about Watson and Crick0132

as being the gentlemen who discovered the structure of DNA.0137

They published a one page paper to the nature of journal in 1953.0143

In their journal, they said that there is this molecule, DNA, that we all know about but0149

it actually has specific base pairing that suggest a possible copying mechanism.0156

This is important because at the time, remember, protein was thought of as being the genetic material.0162

DNA, they did not see any type of copying mechanism.0170

Because at the time, DNA looked as a tetranucleotide with the bases coming off on the ends,0173

the phosphate backbone being in the middle.0183

This would be the phosphate backbone and this would be a planar structure.0187

You would have a tetranucleotide here, and so on and so forth.0192

They just did not have a way that you can replicate DNA.0195

Watson and Crick’s paper, using Rosalyn Franklin's data without her permission,0199

was what allowed and we went to start thinking about this differently.0205

Remember, what DNA looks like is this, with the phosphate backbone on the outside and the DNA base pairs in the middle.0208

To talk about DNA replication, we have to start somewhere, why not start with initiation.0226

Initiation of replication requires DNTP.0234

DNTP’s are deoxynucleoside tryphosphate. This just means, it is your DATP’s, DTTP, DCTP, and DGTP.0237

What it says is that, what this slide is, is that not only do we have to have those nucleotides,0257

but we have to have a primer template junction.0263

What exactly is a primer template junction?0266

Let me draw this for you.0268

If we did DNA, say that is our 3 prime and 5 prime, this is DNA.0270

Let us say that we have, this is our template and here we have our primer.0282

I will draw it out like this.0291

This is going to be 5 prime of our RNA, this would be the 3 prime.0292

It has an OH, that is our 3 prime.0303

What we need is this primer template junction so that the incoming nucleotide can join at this OH.0308

What really happens is that we have the 3 prime OH is attacking the incoming triphosphide, the incoming DNTP.0318

What it does is, it attaches the triphosphate causing a pyrophosphate which is 2 phosphates to leave.0330

The, that pyrophosphate gets cleaved again, by an enzyme called pyrophosphatase.0340

The reason I'm telling you all of this is that, when you are building molecules,0345

when you are doing anabolic reactions, it needs a lot of energy.0350

We have these GTP’s, ATP's, TTP’s, CTP’s, coming in, in the triphosphate form.0355

They are actually only adding into the new molecule in a monophosphate form.0362

Because, these two phosphates, the pyrophosphate group is being cleaved off.0367

When pyrophosphatase breaks this pyrophosphate into individual phosphates, that gives you a high amount of energy.0373

It has a very large negative Δ G reaction and the KEQ is very high meaning it is basically irreversible.0383

It is an irreversible, high energy, giving into the system reaction.0391

This is important, the DNTP, let us say this GTP would come in there and0398

eventually what it would be added as is a DGNP because the pyrophosphate has been removed.0407

That is how we need to start with, we need that primer template junction.0416

Let us even rewind further than that because we need to start somewhere.0422

To get this primer template junction, we need to be able to find where we need to start replicating.0429

That is where we do origin recognition and that is done via the origin recognition complex which you can just call ORC.0437

That is composed of many different proteins, the ORC protein, CDC6 and CDT1, amongst others, these are the main ones.0446

This is a complex of proteins that recognize the proper DNA sequence for initiation of replication.0453

It was also recruit DNA helicases to open up the DNA.0462

This is a little separate from prokaryotic replication, what I told you right now is eukaryotic.0469

Prokaryotic replication will begin at a single unique nucleotide sequence called the ori-site or the origin of replication.0476

What we have happening there is DNA A will bind AT rich sequences.0485

Remember why AT, if we think back, AT has 2 hydrogen bonds, GC has 3 hydrogen bonds.0492

AT rich meaning there are less hydrogen bonds to melt, it does not cost us much energy.0502

DNA A will bind to those AT rich sequences at the ori, and start to melt the DNA.0508

The helicase can come into bind.0516

This is prokaryotic.0518

How I’m running this whole unit right here is that, we will talk about the eukaryotic0520

and then underneath in the bullet points, I will give you the prokaryotic homologous proteins.0526

Here is the initiation of replication.0532

Once again, we have our initiation occurring where we are going to start having something0536

bind at the sites of replication, the origins of replication.0542

It is important to know that our bacteria that are ppp, usually have a circular genome.0549

Our circular genome is only going to have one site, one ori site.0557

Replication is going to occur in both directions until it ends at the termination site down here.0571

Our eukaryotic organisms are going to have many different sites, many different origins of replication throughout the chromosome.0579

You can have anywhere between a separation, maybe 30 kilo bases in between these origins of replication.0597

Based on individual organisms and individual chromosomes,0605

that can still give you anywhere from ten to thousands of origins on a single chromosome.0609

This is eu and this is prokaryotes, this would be one origin.0631

What we have is many origins of replication.0644

We can start, I will just zoom in.0647

What happens is that we have the melting of DNA.0651

This is all still hydrogen bond.0667

This, in the middle right here, has started to melt.0672

The DNA is melting and we are going to start loading on our helicases.0676

Our helicases are actually hexomers, 6 different subunits of proteins, added on and they will move in a certain direction.0686

We will have one at each site, they look like doughnuts.0699

They will be moving in opposite directions.0704

Once again, we always label our polarity of our strands.0707

Our helicases, as I have already alluded to, in eukaryotes0716

those are called MCM proteins or mini-chromosome maintenance complex.0720

What those are, it is a hexomer, a homohexomer.0726

We have 2, 3, 4, 5, 6, 7 subunits, they catalyze a separation of that double stranded DNA helix.0733

What is important is that, as I said, the bond is a double hexomer.0744

They will bind DNA after being loaded onto it in G1 phase, this is important.0750

It is already there, once we move into S phase but they are not active.0756

They have to be activated by certain kinase which is called CDK cyclin-dependent kinase.0765

DDK which is DBF for dependent kinase.0781

Once those are activated, once we get to S phase, then it will start opening up the helix.0795

G1, we are loading the helicases but they are not active.0802

Once they get activated in S phase, then we can go through DNA replication.0806

We have our helicases binding DNA.0813

When they are actually active, they will spin the DNA apart.0817

They will unwind it and they do this by using ATP.0821

For every two bases that it pulls apart, it uses 1 ATP molecule.0826

It is very energy intensive.0832

This helicase is spinning at about 10,000 RPM’S, 10,000 rotations per minute that this helicase is spinning.0836

That is very quickly and this is because we need replication to occur.0849

Synthesis phase, we have 8 to 12 hours but we also have to replicate over 3 billion bases.0853

If we are looking at the prokaryotic side of this, we have DNA B in prokaryotes being the helicase,0862

this is requiring DNA C to bind the DNA.0869

Once we have separated some DNA, we are going to have some double stranded parts of DNA.0880

And then, we have some single stranded parts of DNA.0895

The single strand parts of DNA need to be kept apart.0900

We have proteins called the single stranded binding proteins.0905

In eukaryotes, that those are RPA.0910

RPA, it is just replication protein A.0916

RPA, those proteins are going to bind these single strands.0920

What it does is it not only keeps them from re-hydrogen bonding back to their complementary strands.0930

It protects them from any nuclease activity.0939

Remember, nuclease is what will cut DNA.0943

It usually does so, some of them do so, none specifically.0946

There is nothing to keep a nuclease from coming in and cutting this DNA.0949

If it were to cut it, let us say at two different spots, we can lose a piece of this DNA.0955

That could just go off in space.0961

We have seen these binding proteins, bind on to that DNA not only keeping the two strands apart0963

but preventing it from being broken apart by our nucleases.0970

The protein that does this in eukaryotes is RPA.0978

The protein that does this in prokaryotes is called SSB, single stranded binding protein.0983

As I mentioned in our previous unit, when the helicase unwinds, we need to think of our,0991

back to our old telephone cord, the coil.1003

When you pull from the middle, you create a bubble.1006

In this case, that is our replication bubble.1009

As you pull apart that middle, on either side of the bubble, we will draw that out.1012

On either side of this bubble, we are increasing the supercoiling.1020

That is just increasing the supercoiling.1032

You are getting positive supercoils ahead of the fork.1036

You get negative supercoils behind the replication fork.1041

You are still going to have supercoils here.1048

If we do not relieve the stress of these extra supercoils, we are going to end up breaking our DNA.1051

That is where our topoisomerases comeback in.1058

Topoisomerases are proteins that help relieve that tortional stress, that is exerted on the DNA from the over winding.1061

We have type 1 topoisomerases, that make one strand of the double helix.1070

We have type 2 topoisomerases, that make breaks in both strands.1079

These are both occurring ahead of the DNA helicase.1086

In the direction of the helicase is moving.1094

In bacterial or prokaryotes, it is not topo 1 and topo 2, the protein that does this is DNA gyrase.1097

Remember, MCM, these proteins of the helicase, they get loaded on in G1, they activated in S.1109

They start unwinding, the single stranded binding proteins will come in, RPA.1117

Then, topoisomerases 1 and 2 will start cleaving, with a nuit, whether it will be a single strand,1123

if it is topo 1 or double strands if it is topo 2, being torsional stress.1131

What we have so far, is that helicase is now in the, let us say helicase, we are just going to draw it as a doughnut.1148

Remember, these are still hydrogen bond.1171

Here is our helicase, remember, we have our single stranded binding proteins are going to be bound,1185

until we need other stuff coming in, other machinery.1191

Topoisomerases are nicking, maybe double nick, relieving torsional stress.1196

And then, we can finally have our replicated polymerases coming in to start DNA replication.1206

Now that we set the scene for the story, let us talk about actual replication.1216

We have the synthesis of the RNA primer by DNA polymerase α.1223

DNA polymerase α has two different functions.1230

One is the primase function and that primase is an RNA polymerase that will synthesize an RNA primer de Navo.1236

De Navo is just a Latin phrase meaning from the beginning and that just means we are making it from scratch.1246

We do not need any previous 3 prime hydroxyl to add on to, we can just add it from scratch.1252

Then, we have the second part of the DNA polymerase α which is the DNA polymerase part of that.1262

That is where we actually have the synthesis of DNA nucleotides, instead of the RNA nucleotides.1268

In prokaryotes, we have the primates function being catalyzed by DNA G.1276

What this is going to look like is, is if we have our DNA, for example,1281

we have we will call this 3 prime and we will call that 5 prime.1296

Our DNA polymerase α, as well as our other replicative polymerase that we are going to talk about,1307

always synthesize in a single direction, meaning they have polarity.1312

That direction that they synthesize in is 5 prime to 3 prime.1317

Meaning, they always add the new incoming base to the 3 prime hydroxyl of the previous base.1324

DNA α comes in, it can lay down its RNA primer.1334

That is about 10 to 20 base pairs and it is happening in a 5 prime to 3 prime direction.1343

Then, the DNA polymerase part of our polymerase α will start making DNA.1354

They might do about 20 base pairs, leaving us with the 3 prime that has a free OH.1366

Once again, if we look back, this is our primer template junction now.1377

We have DNA and now we could bring in our replicative polymerases to continue on with that.1382

Why do we even need DNA polymerase α?1391

The reason being, poly-Δ and poly-ε are replicative polymerases, first of all, cannot sympathize de Novo.1397

They would not be able to lay down this first base.1408

They cannot add to an RNA.1413

They need, not only the RNA part, so that we have the primase function laying down the primer.1418

We need this to re-prime hydroxyl, that is a part of the DNA base.1424

We can have the rest of our polymerization synthesis occurring this way.1432

We will talk about that in the next few slides.1437

This whole sequence right here is polymerized or synthesized by DNA polymerase α.1444

This, remember, is the original DNA template strand.1474

Remember always, we want to label our polarities of our DNA strands because1488

that is going to be able to tell us in which direction we are going to synthesize DNA.1493

It is going to be in the 5 prime to 3 prime direction.1497

Let us go to the leading strand.1504

The leading strand is synthesized by DNA polymerase ε.1507

DNA polymerase ε still has a 5 prime to 3 prime directionality but it has a 3 prime to 5 prime exonuclease activity, as well.1513

Which this, we can just think of as an eraser function.1527

It is called proofreading meaning if it has added the wrong base, it can back up and erase it,1532

and then, put in back the correct base.1544

DNA polymerse ε synthesizes the majority of the leading strand.1547

Processivity of this enzyme is increased by its association with PCNA which is the sliding clamp.1554

It is called the proliferating cell nuclear antigen.1564

It is also this PCNA is loaded by RFC which is replication factor C.1569

What is processivity?1578

Processivity, all that means in layman’s term is that,1581

it increases the amount of bases that can be synthesized with each time it interacts with the DNA.1589

Really what it is, it is the enzymes ability to catalyze consecutive reactions without releasing its substrate, which in this case is DNA.1597

The higher the processivity, the more bases we can be synthesized before the enzyme falls off and has to then get back on the DNA.1607

Let us write this out, we have our replication fork opened up.1622

Helicase moving in that direction, we will call this the 5 prime to 3 prime.1642

In black is going to be our parental strand.1652

Remember, what we have is we have the primase function and then the DNA polymerase function.1663

This is all done by poly-α, this will be the 5 prime of that.1677

After poly-α has thrown down about 10 base of primer, about 10 base pairs of DNA,1688

then we get DNA polymerase ε coming in, taking over and synthesizing, and following the helicase.1694

This is called the leading strand because even though it is following,1719

it is actually the one that is going faster and longer than what is called the lagging strand.1725

Another name for the leading strand is the continuous strand.1731

Now that makes more sense when we talk about this.1740

It is continuous meaning we do not have any stops.1743

DNA polymerase ε, theoretically, could continue synthesizing after being primed a single time,1745

all the way to the end of the chromosome.1752

What happens is, as the helicase continues to move toward the right of our screen, it will open up more and more DNA.1757

If we just erase this, when we open up this DNA, we are having the helicase moving even further.1768

As long as the helicase keeps opening up DNA strands, opening up free single stranded DNA,1796

poly-ε is just going to keep polymerizing, keep synthesizing DNA.1806

This is the really simple strand, the leading strand.1814

This, once again, in green, as well as the primer associated with it, is the leading strand.1817

This black part is the parental strand and this is the leading strand template.1824

They are moving in this direction.1841

This would leave us with our 3 prime right here.1843

That is the more simple of the two strands.1846

Now, let us talk about the lagging strand or the discontinuous strand.1849

I already drew out what I wanted to show on this slide.1855

One more time, as the helicase moves, translocates from this area to this area,1860

we are going to open up more and more, allowing the proper synthesis of the leading strand.1876

Once again, always, the polarity.1902

What is our 5 prime to 3 prime exonuclease activity?1917

An example of that would be, if we have a 5 prime to 3 prime sequence of DNA.1922

Let us just say it is AT, GC, AT, GC, all are 5 prime to 3 prime exonuclease activity.1934

Remember, exo means outside or the ends.1944

Nuclease, it is breaking up nucleic acids.1949

All it is, it is just an eraser function from this side.1952

Right there, you just saw 5 prime to 3 prime exonuclease activity.1959

The lagging strand is a little more complicated than the leading strand.1970

The leading strand, once again, is continuous.1977

The lagging strand is what we call discontinuous.1981

DNA polymerase Δ, this is Δ, will synthesize the majority of the lagging strand of DNA.1995

Its processivity is also increased by associating with PCNA which is loaded by RFC.2002

What we see different in the lagging strand than we see in the leading strand, are what are called Okazaki fragments.2011

What is synthesizing these in prokaryotes is DNA polymerase 3 not DNA polymerase Δ, as in eukaryotes.2021

If I can draw this out for you, let us go back to our nice little replication bubble, show you the lagging strand.2035

Here is our helicase, moving in this direction, we have our polarity, this is our parental.2049

I will show you our leading strand.2060

This is poly-ε, this is poly-α.2075

What we have on our leading strand, we are following the helicase.2088

Our lagging strand are going the opposite way of our helicase, meaning we are going in this direction.2094

What we have is, for example, we lay down a primer, we lay down that little piece of DNA all by poly-α.2103

The 5 prime is over here meaning we have to go to the left side.2116

Poly-Δ is what is going to synthesize the majority of this strand, this is poly-Δ.2121

Poly-Δ is usually only going to synthesize about 200 bases at a time, before it falls off.2133

Poly-ε can do thousands or tens of thousands of base pairs at a time, before it might fall off.2143

Theoretically, all you need to do is at a primer to the leading strand one time,2151

you can synthesize all the way to the end of the chromosome, whereas, that cannot happen with the lagging strand.2158

The reason why, it has everything to do with the polarity of the polymerases, as well as the polarity of the helicase.2168

As the helicase keeps going to the right, we will free up more and more single stranded DNA.2180

Look, we freed up enough single stranded DNA, we can throw down another primer.2188

We have poly-α, throwing down a primer, a little bit of that DNA.2194

This is its 5 prime end.2206

And then, we have poly-Δ coming in and go on about 200 base pairs or so.2210

As the helicase is still there, we cannot go any further.2222

We need to wait for more of the single stranded piece to come up our way.2226

If we just say that the helicase keeps going in that direction and is opening up more,2231

let us say it is opened up more, our leading strand that will also be your prime, it keeps going.2238

The lagging strand has got in some opening.2263

Now, we can throw down, let us say another primer go through there.2266

The, we have poly-Δ, this is poly-α.2275

Remember, our 5 prime, this would be the 3 prime end of that.2285

It is the discontinuous strand because it has to wait until helicase2294

frees up more single stranded DNA for a new primer to be thrown down.2297

As you can see, we would have RNA in our DNA complex.2308

We need to process those fragments.2314

In case I forgot to mention, when we have the parental DNA, 5 prime, 3 prime,2319

primer this is our leading strand, this is our lagging strand.2343

Each one of these primer DNA pieces on that lagging strand, that is called the Okazaki fragment.2372

Each one of those, I have written it as having two different Okazaki fragment.2391

Each one of the primer laid down by a poly-α, with a little bit of DNA by the poly-α and2397

the DNA synthesized by poly-Δ, each one of those little pieces of discontinuous fragments are called Okazaki fragment.2405

We have RNA in each one of those, we have a lot of RNA down on the lagging strand.2412

We have just this hopefully, this one piece of RNA on the leading strand.2418

We need to get rid of that RNA, we want only DNA in our DNA double helix, after it has been replicated.2424

How do we go throughout doing this?2432

We have to go through Okazaki fragment processing.2434

Okazaki fragment processing happiness when we have poly-Δ, phen 1, and DNA ligase, all coming into play.2440

If we are talking about happening in prokaryotes, this is carried out by DNA polymerase 1 and DNA ligase.2450

In E coli, replication termination actually occurs when we have this two protein bonding to a ter-site on the DNA.2460

It is a little different, that is why we are focusing on eukaryotes.2468

When we have this Okazaki fragment processing, what we end up having is poly-ε, if we draw it out again.2473

Remember, this is done by poly-ε, this was done by poly-α.2522

This is your leading strand, from here to here, that is your leading strand.2532

This is your leading strand template.2542

This is your leading strand.2556

Up here, we have your lagging strand template.2565

This is helicase, always moving into 5 prime to 3 prime direction.2581

Over here, we have that is a 5 prime.2592

Okazaki fragments, each of these.2613

For processing, what actually occurs is that we have polymerase Δ, actually displacing or going underneath,2643

if we take the arrow ahead of this one.2655

Remember, this is all going to be hydrogen bonded, all these stuff.2659

Poly-Δ, as it is polymerizing an Okazaki fragment, it is going to come under and2673

displace about 1 to 2 base pairs, 1 to 2 nucleotides of this flap, of this primer.2681

What it looks like up close is, normally, here is your DNA, this is your primer.2694

When the polymerase Δ comes in, it actually ends up lifting up this primer and2710

allowing a poly-Δ to come in and continue polymerizing.2725

And then, what we have is phen 1, flapping on nucleus 1 is a protein that comes in and cut the non based paired primer out.2730

It will cut that out, then we got nerves out.2746

What happens again, we have this just continually repeating.2750

DNA polymerase Δ keeps coming in further and further.2757

They are displacing more and more of that RNA.2775

That RNA will pop up again, phen 1 comes in, cleaves, and it is basically left free.2779

That continues to go in, even through maybe some of the DNA bases.2791

What we see over here, if we are watching it, even some of the DNA bases,2796

it is there and what happened is we have erased that RNA primer.2804

That is what we call Okazaki fragment processing or the removal of the Okazaki fragments.2808

We would have to do this for each one of the primer C, even the ones on the leading strand.2815

Once we have that, we just have a nick.2824

We do not have a nick in the backbone, we have a nick in the backbone and it is all that we have.2828

We have then, DNA ligase coming in and sealing that nick2836

by covalently joining that free 5 prime phosphate with the 3 prime hydroxyl.2840

The 5 prime phosphate, the 3 prime hydroxyl, gets sealed together via DNA ligase to seal the nick in the backbone.2853

An example of our 3 prime to 5 prime exonuclease activity, we have talked about before, DNA poly-ε has this.2864

If I were to write out the sequent, AG, TC, AG, TC, 3 prime.2872

All of that is, it is an eraser function, like this, we can erase.2882

That is your 3 prime to 5 prime exonuclease activity.2889

This is important when, let us say bound to, if we write this back out, AGTC.2892

If this is not nearly synthesized, this is TCA, GTT, CAG.2907

This is 3 prime, 5 prime.2919

Let us say that this is the parental and this is the nascent or newly synthesized strand.2922

What if it accidentally put in an A right here?2932

That is the incorrect base pair, noticed G’s with c’s, A’s with T’s are proper.2942

It can use its exonuclease activity to erase that, and then come back and properly put in the C.2947

This is important.2956

For example 3, I want you to draw a replication fork and label the Okazaki fragments, in order of when they are synthesized.2963

We will draw this together, we will do our parental.2974

Helicase always go in that direction.2990

Remember, do not forget your strands polarity.2992

Let us, first of all, do the easy one, the leading strand.3004

Primer with a little bit of DNA, the polymerase comes in and synthesize it all the way behind that helicase.3011

Remember, this is poly-ε and poly-α.3023

Over here, we have the same thing.3031

If I drew, here is a primer, a little bit of DNA, synthesis.3033

Primer, a little bit of DNA, DNA synthesis.3043

Primer, a little bit of DNA, DNA synthesis.3050

Let us not forget.3060

Remember, we have the red is in poly-α, the blue over here is poly-Δ.3066

What I want you to do is label the order in which these Okazaki fragments were synthesized.3080

If we think back to the fact that our Okazaki fragments are discontinuous,3089

our lagging strand is discontinuous, the reason is we have polymerization on this top strand, going leftward.3097

But we have the helicase direction going rightward.3108

We have to wait until the helicase frees up more DNA.3113

The answer to this question is, the most recently synthesized Okazaki fragment is the one closest to the helicase.3117

This one is older and this one is the oldest.3127

So far, I have shown everything in simplicity.3136

his replication fork, they often go in both directions.3139

You would actually have a helicase going in this direction as well.3147

At the midline, we are just going to say this is the midline because I have already drawn over here.3153

At the midline, we actually have a switch of, this is the leading strand, this is the lagging.3164

We still have our, this is our 3 prime and this is our 5 prime.3189

As the helicase is going in the other direction, as if right here,3194

this means we have a switch of the lagging and leading strand.3208

Up here, we will give this nice little delineation of the midline, what we have is,3212

this becomes the leading strand following the helicase.3223

This becomes the lagging strand.3234

Once again, our 5 prime, this would be the 5 prime over here.3251

I have shown you simplicity so far, but this is really what is happening.3265

The replication fork, when it fires, it is going to go in both directions.3268

You are going to have, over here at the top strand, the replication being the lagging strand,3272

the bottom strand being the leading strand.3280

Both when we are working in the other direction, we actually have the top strand being the leading strand3281

and the bottom strand being the lagging strand, the discontinuous strand.3288

Hopefully, this was clear enough for you to see how we have the leading and lagging strands.3292

Let us label these which one was the first or the newest one made.3299

This is the newest one made.3310

Actually, let me delete that and make it a little easy for you.3319

This was actually the first one made, this was the second one made.3325

This was the first one made, this was the second one made.3331

But the one closest to the helicase is the newest.3335

The Okazaki fragment, closest to the helicase is the newest.3339

We have talked about the fact that everything needs to be primed.3363

We have talked about Okazaki fragment processing.3367

We do not talk about what happened until we get to the very ends of the chromosomes, at the telomeres.3370

What is a telomere, first and foremost, and why does it bring about what is called the end replication problem?3377

Telomeres are regions of repetitive nucleotide sequences.3385

In humans, that sequence is TTA, GGG.3390

That is found at the end of each chromatin.3398

If we see down here at the picture, the things loop up in white, at the ends of each chromosome,3400

those are our telomere sequences.3411

Telomeres possess a 3 prime over hang.3416

The reason being, when we go through and take off that primer, if we look back here is our DNA.3421

The end of our chromosome, this is 3 prime, 5 prime, 3 prime, 5 prime.3431

This, remember, was a primer.3446

When it gets removed, we have a little bit shorter 5 prime end on the very ends of our chromosomes.3451

We have a 3 prime over hang.3460

This is important, we will talk about it in the next slide.3462

Telomeres will act as caps for chromosomes.3465

They do that by binding a bunch of proteins.3469

These proteins help for a couple of different things that I have mentioned before.3472

First of all, it protect the end of the chromosome from exonuclease.3476

Remember, exonuclease could just come in here and start erasing, keep erasing.3480

If we had a gene here, if this is gene sequence, if we keep erasing with nuclease,3489

we are going to get into a gene and you are going to affect that gene.3500

That can be big time problems, it can either lead to apoptosis which is okay, the natural death of a cell, or it can lead to cancer cells.3503

Other than protecting from exonucleases, these telomeres, by binding proteins,3513

they protect from fusion with other chromosomes.3520

Fusion of a couple chromosomes together will affect how they get segregated into daughter cells,3525

whether it would be mitosis or meiosis, this can affect how many chromosomes you get.3531

You can have what is called, for example, let us say trisomy 21, that can lead to mental retardation.3537

There other types of fusions and we are most likely talking about fusions, strike that,3544

take out the trisomy 21 part, that is just an extra chromosome.3553

When we are talking about fusion with other chromosomes, we can have chromosomes breaking3561

or just binding together via their end regions, their telomeres.3565

That will make it to where certain cells do not even have, let us say for example, a chromosome number 20.3570

The other chromosome, the 20 that should have been in that cell is in another cell, that still has two chromosome 20 per cell.3582

A lot of these can cause cancer.3591

Telomeres, in the replication problem that I have talked about before,3597

the fact that we have to use a primer to initiate DNA synthesis is followed by the removal of that primer.3603

We obviously have to take out that primer.3612

We do not want RNA in our DNA, that causes the progressive shortening of the DNA after each replication cycle.3614

If we draw our telomeres again, we have the 5 prime, 3 prime, this was a primer that we have to remove.3624

We have a 3 prime over hang.3645

If I go like this, if I'm going to replicate again, after one replication, this one is going to be even shorter.3648

If this was the template, we are only able to synthesize 5 prime to 3 prime, 3 prime to 5 prime.3673

This ends up getting shorter and shorter.3690

And then, we can do that even more.3705

This is after it is all been processed.3734

As we can see, the chromosome used to be to here.3736

We have gotten shorter with each replication, and shorter and shorter.3745

At which point, we can eventually end up inside of a gene.3755

If we start deleting parts of our genes, as I said before, we are either going to apoptosis or one might end up becoming a cancer cell.3761

This causes the progressive shortening.3778

In most eukaryotes, we have the ends of this linear DNA, our telomeres,3781

being replicated by unique mechanism that we call, using the enzyme called telomerase.3787

Telomerase is that protein that can help alleviate this end replication problem.3795

It is a ribonucleic protein that means it consists of an RNA, as well as protein.3808

It uses this small RNA molecule as a template for reverse transcription.3816

Remember, reverse transcription is making DNA from RNA.3821

It uses complimentarity between its RNA template and the telomere DNA sequence, and that binds the DNA.3825

It will eventually extend the 3 prime OH of DNA, by reverse transcription.3834

They will extend it, the will translocate, extend it more, translocate, extend it more.3841

DNA polymerase α will come in and synthesize the new DNA, using this new longer 3 prime end as a template.3849

This is happen in here.3861

We have the binding here, we have the extension here.3870

This is just another way to look at telomerase.3880

Telomerase as a factory, this the protein part of it, here is the RNA part of it.3882

This is complementary to the telomeres sequence of whatever organism, in this case humans.3887

It binds to that, then we have a synthesis, we have an extension of that 3 prime end.3897

Eventually, we can have a polymerase come in, be primed by that DNA polymerase α and extend 5 prime to 3 prime, as always.3903

The last example for this unit, I want to mention reverse transcription because I talked about it before.3923

Remember, the central dogma, DNA, RNA, protein.3928

DNA to DNA is replication, which we talked about today.3940

DNA to RNA, transcription.3949

RNA to protein, translation.3958

Reverse transcriptase, that is an enzyme that is capable of doing reverse transcription.3965

What that does is, it turns RNA into DNA, just like telomerase.3974

Examples of enzymes that have reverse transcriptase capability, probably, the famous one is HIV.3989

Another one is the hepatitis B virus, they both contain reverse transcriptase enzymes,3997

if we talk about any of the viruses in the retrovirus family.4005

One last thing I want to talk to you about telomerase.4015

We have it usually only being active in germ line and stem cells.4019

It is reactivated however when you mobilize cells.4025

A mobilize cell, that is using code word for cancer.4029

Our cells are supposed to eventually die.4036

As the telomere shortened, that is somewhat may be correlated with aging.4039

Telomerase may regulate aging and senescence, that just means the not dividing, basically, your G0 phase.4045

It does that by increasing what is called the hayflick limit.4055

The hayflick limit is the measure of the number of times a normal human cell population will divide,4059

before cell division stops and it undergoes senescence.4067

This is usually regulated by getting to that critical limit of the telomere.4072

If you increase telomerase, in cells, if you increase the activity, you can lengthen the telomeres and4078

decrease the critical lengthening limit.4086

Therefore, increasing the hayflick limit.4089

However, we do not want cells to be able to divide forever.4091

When cells can divide forever, they become cancerous because they often grab more and more mutation.4096

In a lifetime of a cell, they get mutated over and over again.4104

As they start to power out and as the mutations get into genes, that causes the cell to go haywire and become cancerous.4108

We do not want that.4119

Some cells, instead of having telomerase activity, they have a different way of lengthening4121

their telomeres called alternative lengthening of telomeres.4129

That will occur into some telomerase negative cells, as well as some tumors.4133

This is where we have the 3 prime end of the DNA forming what are called T loop structures.4138

They use the process of strand invasion which we are going to talk about later in lecture 8 or 9,4146

when we talk about homologous recombination.4153

It uses strand invasion, to use one strand as a template for new DNA synthesis.4156

This is important because, we cannot always use telomerase inhibitors as a way to kill cancer cells,4169

because some cancer cells have found a way around it, by using this.4177

That is something that is always having to be looked into.4182

I will leave you at that, I would like to invite you back.4186

Thank you for visiting www.educator.com, please make sure you come back and see me again.4192