Molecular Biology Homologous Recombination & Site-Specific Recombination of DNA

Hi, and welcome back to www.educator.com.0000

Today, we are going to talk about homologous recombination and site-specific recombination of DNA.0002

This is linking back on the previous unit where we introduced the concept of homologous recombination.0007

Now, we are really going to get into the details.0013

As an overview, we are going to talk about all things, homologous recombination.0017

Double Holliday junctions, resolution vs. the synthesis dependent strand annealing, which we introduced last time.0023

And then, we are also going to talk about single strand annealing, break induced replication, meiotic recombination,0032

as well as double strand break repair in prokaryotes.0038

Finally, we will have a short overview on site-specific recombination.0042

Homologous recombination, once again remember, we can call this HR, is genetic recombination which the nucleotide sequences are exchanged0051

between two similar which are called homologous or identical molecules of DNA.0061

Two similar molecules of DNA are called homologous DNA.0068

This will produce new combinations of DNA sequences during meiosis.0073

HR is often used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.0078

That is called horizontal gene transfer, it is the passing of DNA from one bacteria to the other bacteria.0088

As opposed to the passage through the new generations of proliferation.0098

Homologous recombination that occurs during DNA repair can result in what is called a non-crossover product.0107

Meaning, it is fully repaired back to normal as if nothing had happened.0115

We are also going to talk about forms of homologous recombination where we get what are called crossover products.0121

Homologous recombination, reminders from last unit.0130

It repairs double strand breaks during S phase or G2 phase using long homologous sequences, as templates for synthesis.0137

If we look here, the red and blue chromosomes are called homologs or homologous chromosomes.0145

They are not the exact same sequence but they are very similar.0157

The process of homologous recombination encounters several of these things that we are going to talk about.0164

Generally, we start with resection, we then go to strand invasion, followed by Holliday junction formation.0172

And then, either double strand break repair or synthesis depended strand annealing.0183

After a double strand break occurs, the MRN complex will bind to DNA on either side of that break.0199

We then have 5 prime to 3 prime resection which allows us to have a 3 prime overhang.0210

And then, we have proteins coming in, binding these 3 prime overhangs, specifically RPN RAD51, we are talking about eukaryotic right now.0218

This forms a nuclear protein filament.0228

What that means is it is a filament of nucleic acid, our DNA and proteins, our RPA, RAD51, and others.0231

We then go through what is called homology searching.0239

We are looking for a piece of DNA that has roughly the same sequent, and then,0242

we go through a process called strand invasion.0248

That nuclear protein filament invades the other homologous chromosome.0251

It goes into that DNA duplex.0258

If we are talking about being in mitosis, you would be invading a sister chromatid.0261

Those are identical chromosomes, that is the best time that you can do this.0268

Or if we are in meiosis, you are using homologous chromosome.0271

It is not the exact sequence but basically the same, it is very similar.0274

When you go to strand invasion, you form what is called a displacement loop or a D loop.0279

DNA polymerase, once we got through strand invasion,0289

DNA polymerase will extend the end of the invading 3 prime strand via normal DNA synthesis.0292

It is using the new DNA from that homologous chromosome as a template molecule.0300

This whole strand invasion and initiation of synthesis will form a cross shaped structure which we call a Holliday junction.0308

DNA synthesis will continue and then that is continuing on the invading strand,0323

the original piece of DNA not the homologous new piece, effectively actively restoring the strand on the homologous chromosome0330

that was just placed during the strand invasion.0338

The double strand break repair is then completed by one of the two pathways.0342

We either have the double strand break repair which we can also call the double Holliday junction model.0346

Or SDSA which is the synthesis dependent strand annealing model.0354

Let us talk about the first one being the double Holliday junction model.0364

The double strand break repair pathway is unique, and that the second 3 prime overhang0372

which is not involved in strand invasion, will also form a Holliday junction with the homologous chromosome.0377

Now, we have what is called the double Holliday junction0384

that will then get converted into recombination products by endonucleases.0386

We have cleavage events occurring here.0393

The double strand break pathway often results in crossovers.0396

This is the model of how a crossover of homologous recombination occurs during meiosis.0406

We will talk specifically about crossing over during meiosis in a few slides, after we have explained these models.0414

For example 1, back to this picture again.0423

What are the combinations in the Holliday junction pathway in that model, results in chromosomal crossover or not,0427

is determined by how the double Holliday junction gets resolved.0436

Chromosomal crossover will occur if one Holliday junction, let us call them the crossing strand and the other on the non crossing strand.0442

What that means is, if we look down here, this is the double strand break repair side.0449

If we cut at the purple here and the purple here, we will get a non-crossover product.0456

If we cut at the orange arrows here and the orange arrows here, we will also not get any type of crossover.0475

If we cut at the orange arrow at this first Holliday junction and the purple arrow at the second one, we will get a crossover, and vice versa.0488

Same thing, if we cut at the purple arrow here and the orange arrow here, we will get a crossover.0500

If we cut at both purples or both oranges, no crossover.0508

If we cut at one orange, one purple, we will have a cross over.0514

Having a crossover is more common than having a non-crossover product.0520

Let us draw this out, so we can see what is going on.0529

We will use different colors so we can see the different chromosomes.0536

First things first, we have a break in the chromosome.0546

What I’m going to do to keep track of what our DNA has is, I'm going to label 3 different low side, that is just a piece of region of DNA.0563

I’m going to say that this is region A, locus A, locus B, and locus D.0574

The homologous chromosome is going to be unbroken.0586

We are going to label these with a, so as to differentiate them, all lowercase.0598

First things first, after that break, we go through resection.0608

We are not going to mess with the homologous chromosome because there is nothing wrong with that one.0633

What we have had here is resection 5 prime the 3 prime, that leaves us with some overhangs, it is a 3 prime overhangs.0686

We can go through strand invasion.0702

Let us continue on down here.0724

What we have, we have our 3 prime, we have our 5 prime, we have both a that have been resected.0728

We have b and d, we have a.0743

What we are going to do, let us draw out this one.0755

What happens here is that, this strand starts to invade and pop up, it pops up this top 5 prime strand up here.0780

It moves this up.0804

This right here, this dotted line, that is all new synthesis that is going to be occurring.0814

This is the strand invasion process happening.0822

We are going to start synthesizing this way.0826

What we are going to be doing, since we are synthesizing using this bottom strand as the template,0834

remember the top strand up here, it was BB.0840

What we are doing this is, since we are using this is a template, this is actually being made as a b, that locus.0844

It is matching the homologous chromosome not the original chromosome.0852

For example, let us just say what this B vs. b could be.0858

Let us say that B that we are seeing, let us say that is an AT base pair and let us say that b is a GC base pair.0865

That might make it more easily understandable.0876

There is the start of our strand invasion.0882

What we can proceed with is the full double Holliday model, double strand break repair.0887

This is what we are going to have.0910

What is happening here is that, this is being synthesized using this piece of red as the template.1010

What we can do is if we call the crossing points, this is where we are going to have to resect in.1028

If we say that this, we are going to cut here and let us do it in different color so we do not get confused.1042

We can resolve it by cleaving there, in those places.1059

Here is what we can do.1081

What we are going to do is go through resolution.1095

If we resolve this, we have separated it.1104

If we resolve it at both green and green, or purple and purple, this is what we will get.1109

This is at both green or both purple arrows.1198

If we do this at one green, one purple.1219

Let us say the first one green, the second one purple, or vice versa.1232

The first one purple, the second one green, this is what we will get.1236

This right here are considered non-crossover.1288

These down here are considered crossover.1299

Why, first of all, what we are looking at is a, locus A with respect to D.1306

The original molecule, A is found with D, a found with d.1316

That is what we consider as normal or wild type.1325

After all of the finishing of this, the resolution, we see that A is still on the same chromosome as A with D and a with d.1329

We are not looking at the middle right now.1344

That is, we do not have a crossover.1348

Down here, we see that A is now with d.1350

A is with D, A with respect to D, there has been a crossover.1356

Before I mention something that you might have already looked at,1370

right here, we are going to keep this for one more second.1379

As you can see, we have B and b.1389

First, how do I get these, how do I get that from this.1392

Let us show you, 3 prime, A, B, D, 5 prime.1401

Let us show you how we get this.1470

The easiest way to look at is what if I cut it in both purples?1473

What that is basically saying is, what I’m going to do is, if I cut right here,1478

it is basically likely erasing, connecting these right here, connecting that right there.1484

You erase this, connect it right there, connect it right there.1492

What we are seeing is this being A, B, D, A, b, D.1496

That is what is happening over there.1511

What is happening down here, the bottom strand is not touched though, just like the top strand is not touched.1513

We have a, b, d.1519

What do we have when we follow it?1521

We can just follow it, a follow up, b follow up that, d.1524

That is the same thing we get over there.1533

What if we cut above the greens, what is that look like?1563

What we can think of is we are not touching the inner strands at all.1572

What we are doing is, when we make the cuts here, we are cutting both strands.1577

Cut there, cut there, cut there, cut there.1583

We are just making a big X.1588

This down to the bottom and up to the top, this one, up to the top, all the way over, down to the bottom.1590

All you have to do is follow along.1603

What we can see is we have A, A down to b up to D.1606

The other strand is A, b, no movement, D.1618

Over here, a, b, d, no change.1629

Up here, a, B, down to d.1635

A with respect to D, we still have no change.1643

You might get a little difference between the locus in the middle at B,1647

but there is no change A with respect to D, because A is still with D, a still with d.1650

When we cross, when we do one of each, we are going to see something different.1659

Let us say we cut here and here.1688

What is that going to look like, once again, it is just like you erased, you complete those.1703

One here, we have cut here, you just make the X.1711

What is it look like?1722

Top strand A, B, we will follow it in black.1724

A, B, down to d.1731

We can already see that A with respect to D is different.1737

This next one, we have A, b, d, so A with respect to d crossover.1742

Down here, a, b, up to D, so a with respect to D crossover.1753

Down here, a, b, all the way up to D.1764

Sorry, the previous strand, this is a, b, still D, but it is the bottom D.1772

On both strands, a with respect to d has had a crossover.1779

We are looking down here.1783

The last thing that I pointed out and I said let us take a look at this.1789

With each of these, we can have what is called a hetero duplex.1792

That is, when we have, let us say this strand is B, maybe it is a T up here.1798

This is a C, just for example.1807

That has to be repaired.1811

If the hetero duplex is repaired by mismatch repair, back to the original,1813

let us say in this case, it is supposed to be B, B, then everything is okay.1820

See down here, it is B, B, everything is okay.1829

B, B, up here, everything is okay.1834

But down here, what if I say that this is repaired the opposite way.1835

Let us say over here, this is repaired to b, b.1842

That is what we call gene conversion because it was supposed to be B, it should have been AT, but now it is a GC bond.1849

That is going to lead to what we call LOH or loss of heterozygocity.1862

Remember, right here, originally, it was an AT pair and this chromosome was a GC pair,1868

that you have heterozygocity at the B allele.1879

But if they are both repaired back to an AT or a GC, you lose the hetrozygocity,1883

you are homozygous at that allele and that could be problematic.1891

It can lead to disease, for example, this can occur when you have a loss of hetorozygocity at a certain locus,1896

that can lead to something called retinoblastoma which is a type of cancer that affects the eye, the retina.1906

This can be highly problematic.1916

We talked about the Holliday junction model.1924

Now, let us say the other type of double strand break repair.1926

We have synthesis dependent strand annealing which is SDSA.1932

Homologous recombination via the SDSA pathway occurs in the cells that divide through mitosis and meiosis.1939

This will always result in non-crossover products, repaired back to as if nothing ever happened.1946

Whereas, homologous recombination via the double Holliday junction model, more often results in crossover products.1953

Let us talk this through, we have the Holliday junction being formed,1964

just as if it were the other way as well but instead it is resolved via branch migration.1968

Now the invading 3 prime strand gets extended along the homologous, the recipient DNA duplex.1975

This is done via DNA polymerase.1984

It gets released as the Holliday junction slides.1988

We call them branch migration, it is sliding, it is moving.1992

The newly synthesized 3 prime end of the invading strand, remember, is that nuclear protein filament, nucleic acid proteins.1997

That can then anneal some base pair to the other 3 prime overhang in the damage chromosome, through complimentary base pairing.2007

After annealing, we can cleave off some small flaps of DNA.2019

However, this is not resolved, the Holliday junction is not resolved via cleavage, like the Holliday junction model is.2023

This one is resolved by moving and basically pulling back up the strand.2032

I will draw that out for you in the next slide.2036

Here is an example, let us show our SDSA.2042

Here is our 3 prime, we still our A, we have a break.2050

Here is our B and D, A, A, B, D.2057

By the way, on both of these, I have just said that this is a double strand break here.2079

Anything that is missing there, if it is a part of a gene, that is going to break that gene.2085

If you add any part of the missing gene, you are going to have problems down the line in transcription and translation.2090

As I have written in here, I’m saying that it is not an important piece of DNA.2095

Just like the Holliday junction model, after double strand break, there is still the resection.2103

Remember, this is SDSA, we have resection and we have 3 prime.2110

Remember, right here, nothing is happening on the homologous chromosome.2160

We once again have strand invasion.2180

We always want to get our polarity, right.2186

We know which way we are going to be synthesizing.2187

There is strand invasion.2255

Now, we get to the process of branch migration.2258

What is happening there is, what we are seeing, let us draw this out.2270

This is being synthesized right.2313

This is moving in that direction.2341

The Holliday junctions are just moving and then what we end up with is,2346

instead of cleavage at these spots, we have what is called dissolution.2355

No cleavage.2372

What we are basically doing is we are pulling this blue strand, it was pulling it up and pulling it up back to its own chromosome.2380

It is pulling this red one back down to its proper complimentary strand.2392

What we end up having is, if we follow it, we have our A, B, and our D.2398

We are just following all the way across this line.2419

This one, since we are pulling up, we are going to have A, b, D, because it is just going to pull that back up.2422

A with respect to D, no crossover.2439

Let us look at the homologous chromosome to check it.2443

We have the bottom one, it is the easy one.2448

a, b, d, it did not have to worry about anything.2451

This one, remember, we are just going to pull it down.2457

a, b, d, there is not going to be any cleavage.2460

These are a non-crossover.2471

However, let us still look, we do, the original damaged strand, we have a hetero duplex.2479

This hetero duplex still has to be repaired via MMR.2489

If it is repaired back to the BB, it is like nothing ever happened.2496

If it is repaired to bb, we have gene conversion and loss of heterozygocity.2502

Remember, instead of cleavage, SDSA goes through dissolution and that is where we have the 5 prime blue strand,2513

just pulling back up and becoming the template for the 3 prime blue strand.2520

We do the synthesis here right, then, it comes back up.2526

This synthesis, it is just coming back off of the blue strand, not on the red strand,2532

as seen in homologous recombination via double Holliday junction.2539

Once again, we can still have that hetero duplex and that can be affected,2544

whether we have gene conversion or not, based on how mismatch repair repairs that.2548

Those are the two big boys when we talk about homologous recombination.2559

There are other types of homologous recombination.2563

We are going to go a few of them, starting with single strand annealing.2568

The SSA pathway repairs double strand breaks between two repeat sequences and this is important.2571

The sequences have to be repeat.2580

This does not require any type of homology between two different homologous chromosomes or identical chromosomes.2584

This is using just a single double stranded helix.2595

This is unlike the Holliday junction or SDSA pathways.2601

This only requires that single DNA duplex and uses repeat sequences for the repair.2605

Those repeat sequences need to be at least 30 base pairs long.2613

This is considered extremely mutagenic because it results in large lesions of DNA as you are searching for the repeats.2620

I will show you an example of this in just a couple of slides, once I finish talking about SSA.2631

If we are going to talk about specifics, as DNA around the double stranded break gets resected,2642

the single stranded 3 prime overhangs are coated with RPA protein.2647

Remember, RPA is going to help protect this from endonucleases, as well as,2655

help form that nuclear protein filament that can go through strand invasion.2662

RAD52 is another protein that would bind each of these repeat sequences on either side of the break2668

and align them so that they can anneal.2674

After they anneal, their leftover non-homologous flaps of these 3 prime overhangs get cut away by endonucleases and then,2677

new DNA synthesis will fill in the gaps and DNA ligase will ligate those gaps making a new continuous strand.2687

The DNA between the repeat that bring off these flaps are always lost, as these one of the two repeat.2695

This is highly mutagenic because you lose a lot of sequent.2703

Let us see what that will look like.2707

We have our 5 prime to 3 prime resection, our homology search, our annealing in those overhangs,2712

the cleavage of the flaps, and then synthesis, and ligation, to finish it.2718

Let us see what this looks like.2722

Here is our strand, these are regions of homology.2732

We get a double strand break.2743

Those are the repeats.2765

First things first, we undergo resection to find that homology.2768

We are going to resect, we will continue on this line, 5 prime to 3 prime resection.2779

It is basically like your eraser.2792

Let us make sure we still know that we have the repeats right here.2809

The resection in the 5 prime to 3 prime manner, it is basically just doing this, it is an erase function.2816

I will draw this back.2827

The next step will be single strand annealing.2831

We find that we do the homology search, we are looking for these red spots, and then, we do the annealing of those overhangs.2839

What it looks like is this.2847

This is going to be single strand annealing.2853

What it looks like is this.2867

What you are doing is you are just moving this close in that direction.2895

You are pushing them together until these repeat sequences overlap.2901

This right here would be the two of them.2906

You are going to lose one of them so that you can make.2909

You will lose the bottom half of this piece and the top half of that piece to make one together.2914

There is the single strand annealing.2922

The next step, we are going to go through cleavage.2929

We have to cleave those flaps.2936

What we are doing, what we have done here is we have nicked it to cut it off.2940

And then finally, we go through the DNA synthesis and ligation to close the gaps.2985

Here is that one repeat, we have lost one of the repeats.3014

As well as, let us say for example, what we have ended up losing if we compare this to the original,3019

we have lost everything from here to here.3052

We have lost a lot of sequent, that all gets lost.3060

It is better to lose that and be somewhat mutagenic, than for you to have to completely lose this piece of chromosome,3065

however, not our preferred mechanism of repair.3077

Another form of homologous recombination is called break induce replication or BIR.3087

This pathway repairs double strand breaks encountered at replication forks.3094

This is because DNA helicase is trying to unwind the template strand and it leads to find the double strand break.3101

The exact mechanisms of BIR are not exactly clear.3110

There are several proposed mechanisms.3114

3 of them all have strand invasion as the first step, but then they differ in how the deal with migrates,3116

as well as some of the subsequent event.3124

But we do know that BIR pathway is actually very useful in maintaining the length of telomeres,3128

when telomerase, the enzyme, is either inactive or not present in certain cells.3135

When we talk about meiotic recombination, remember, homologous recombination is what is occurring.3148

In meiosis, homologous recombination is required of a proper chromosome alignment and segregation.3154

In meiosis, double Holliday junction always get resolve this crossover.3162

If we think about our homologous chromosomes during meiosis, we have homologous chromosome.3166

When they come together, they will form what is called a chiasma.3181

Right here, this is called a chiasma.3214

This helps a cell go through proper chromosomal segregation.3223

Without chiasma, you are actually increasing, by far, your occurrence of what is called non-disjunction.3229

That is when you have, let say both homologous chromosomes ending up in the same sperm or egg cell.3244

And then, one egg cell being without that complete set of genes.3252

Let us say for example, one sperm would get two chromosome 21, whereas, another cell will get 0 of them.3258

If this chromosome 21 sperm cell fertilizes the egg cell that was produced normally,3273

it has one copy of every gene or one copy of every chromosome,3282

but this sperm has one copy of every chromosome but two chromosome 21.3287

If these guys fuse, you are going to produce a fertilized egg with 3, it is a diploid cell,3292

but instead of two chromosome 21, it has 3 chromosome 21.3304

It is what we call trisomy 21 that may lead to Down syndrome.3309

Chiasma are very important.3321

In a chiasma, we are undergoing crossing over.3324

We are crossing over genetic material.3327

This black one, when we resolve this, what this looks like is that we have a little bit of the red.3329

This one has a little bit of the black.3352

Not only does it help us with non-disjunction events, but we actually have some gene transfer3354

which helps with the variability of the genetic sequence and can help with evolution.3360

This will always how we resolve this in meiotic recombination, this will always result in a crossover product.3372

In meiotic recombination, we can talk a little bit of a detail.3392

We have this protein called SPO11.3395

It is going to make a double strand break at what is called a recombination hot spots.3399

It is a place where it is likely to occur.3404

We then resect, we cut it by using our MRN complex.3409

Here is SPO11 occurring, here is the resection.3418

We have RAD51 and DMC1 as the proteins that coat the single stranded DNA and help with the strand invasion.3424

These are involved in making the nuclear protein filament and making the Holliday junction.3431

This is occurring in here, from the Holliday juncture migration can result in hetero duplex DNA containing mismatches.3432

We are coming down here.3452

The gene conversion can result in hetero duplex that we have talked about before.3456

We can have a loss of hetorozygocity.3461

Instead of BB on one strand, bb on one strand, maybe we have both of them being resolved to all b.3464

You will have all one way vs. none the second way.3472

We are resolving our crossovers.3480

We have already talked about having it resolved.3486

That was just a quick overview of just another few specific proteins involved in the meiotic recombination.3488

We have only talked about eukaryotes, so far.3496

We have to talk just a little bit about double strand break repair in prokaryotes.3499

There are pathways called the recBCD pathway.3503

There is basically just one pathway that they will go through.3508

What happens is that, we have this recBCD protein complex binding to and unwinding our double stranded DNA end.3512

That results, whether they are still binding here, it unwinds creating single stranded ends, as well as a single stranded loop.3522

The two tails will then anneal, re-anneal, to produce our second loop.3536

This is loop 1, this is loop 2.3546

Both loops can move and get bigger.3550

What we then have is recA is going to be binding.3553

It is going to be binding to the DNA, to protect the single strand.3559

We do not want it to get nicked by an endonuclease.3565

We have recBCD coming in, adding on.3571

It is already there, but now we are going to actually cause a nick.3577

We are going to nick this 3 prime strand at what is called the chi sequence.3582

This is just a hot spot for where we have nicks from the recBCD complex.3589

We then unwind further, and then we have what we see as this really long 3 prime end with the chi sequence that it is in.3594

This chi sequence right here, if we follow it around the back.3611

Here is the chi sequence, that is just a hotspot of where we are going to nick.3616

If we continue on to the next side, we have already shown that recBCD loads recA, that is down here.3620

Once recA is loaded, recBCD can disassemble.3630

It comes off of the DNA.3634

The recA promotes the strand invasion in the homologous DNA duplex,3636

producing our D loop, the displacement loop.3643

And then, the D loop gets cut and anneals with our gap in the DNA.3649

This is happening right here.3654

And then, we can resolve this DNA, this complex, this Holliday junction complex via the ruv AB complex, that is going to bind.3656

The ruv AB is going to see this Holliday junction and come and bind.3670

That is then going to recruit ruv C and we are going to resolve it.3676

This is all occurring here as well.3683

This is our final product.3685

That is how prokaryotes would do this, or e coli, specifically.3688

We did all the details in eukaryotes, then we zoomed over the prokaryotes a little bit.3692

This is the overview of how prokaryotic homologous recombination repair occurs.3700

To compare our prokaryotic and eukaryotic recombination.3710

The pairing of our homologous DNA, that is recA in E. Coli.3714

RAD51 and dcm1 in eukaryotes, dcm1 specifically in meiosis.3720

When we are generating that single stranded DNA for strand invasion, that is recBCD in E. Coli or MRN complex in eukaryotes.3726

MRX is used in human.3736

And then, Holliday junction recognition branch migration and resolution,3740

that is the ruv AB complex and ruv C for resolution for E. Coli.3745

In eukaryotes, we have a much more complex set of proteins handling that.3750

That is all of our homologous recombination.3763

There is a different type of recombination.3768

Instead of doing a whole long unit on that, I just added a few slides in this unit just to keep it all in recombination.3770

We have what is called site-specific recombination.3781

There are two types that we are going to talk about.3786

One is called conservative site-specific recombination.3788

That is involving protein enzymes called recombinases.3795

They act really similar to topoisomerases.3800

They cleave and rejoin DNA molecules to either invert DNA fragment.3805

They turn it upside down and flip the sequence of events or to insert them into a new site.3814

You either take it from one side and move it to the other.3822

We also have what is called transposition.3825

This uses a different type of enzyme called a transposase.3829

This transposase enzyme will cleave two ends, other transposable element.3834

It will randomly insert it into another place on DNA which we call the target site.3840

What are these going to look like?3853

Let us talk about our transposons first.3855

A transposases, we cleave both ends of the transposon in the original site and catalyzes integration in the random target site.3861

We saw them on previous slide.3869

We can do this in what I like to call a cut and paste mode or a copy and paste mode.3872

The difference between that is such.3878

Here is double stranded DNA and here is our transposon or a transposable element.3884

We have two options, we have the cut and paste option.3898

That is this, that is where we remove it from here.3905

It was here, we have cut it out of the DNA and now we have moved it to another spot on DNA which is right here.3913

That is what I call the cut and paste version.3925

We can also have the copy and paste which as you can probably already imagine.3935

We leave the original piece of DNA that sometimes we call these jumping genes transposons.3944

We leave the original one and we copy it, and add a second one in the new site.3955

We started with one, the cut and paste we still only have a one.3962

In the copy and paste, now we have two.3968

As we see down here, more than 40% of the entire human genome is composed of repeated sequences.3976

It is very likely that a transposon got into our ancient ancestors.3990

Throughout evolution, throughout the time, it has not only cut and pasted itself but more often,3999

more likely, copied and pasted itself making those repeat show up in many different parts of our genome.4007

Meaning that we have also lengthen our genome, as we have grown as well.4016

The transposons are very likely responsible for a large portion of these repeated sequence.4020

We have a lot of repeated sequence in our genome.4027

I want to give you an example of our site-specific recombinases.4036

Here is an example, what if we have a circular piece of DNA.4042

It wants to recombine with a linear piece of DNA.4054

This can sometimes happen when a bacterial plasmid, a vector, is going to join the into the main genome.4060

What is important here is that we have to see these recombinases work with a polarity.4073

This arrow that I’m showing is not just for no reason.4086

This means that they have the same polarity.4091

What you can do is to insert into this, A will cross like this.4093

We will cross like this.4112

What we end up with, we just follow the polarity, this is actually pretty easy.4114

We can start at X, we have a rightward arrow, and then we follow it out.4122

It goes to B, it comes all the way around.4136

DNA sequence to A, and then A crosses back down, rightward arrow, and then we get to Y.4142

This could be one like, nice long linear sequent from a circular sequence and a small linear sequence.4152

This is what we call insertion.4158

We have inserted this piece of DNA, the circular piece, into this linear piece of DNA.4168

There is also what is called inversion.4175

What we would have here is, if we start with X, rightward arrow to A, long piece of DNA B.4181

We have a different arrow, a leftward arrow to Y.4200

These polarities have to match up.4203

What has to happen to this, we actually have to curve this around.4205

Because look, the arrows pointing at B so it is always going to be pointing at B.4223

How we do this, we are going to once again make a nice X.4228

We can start with X.4237

We are going to go this way.4240

What we have, we start with X, we have a rightward arrow.4245

We come to B, follow it all the way around to A, this long piece of DNA to A.4254

What do we have, we have an arrow that is going to be pointing at A.4262

A leftward pointing arrow and then down to Y.4270

This is what we call inversion.4276

This one we can flip a DNA sequent, as we see here, look what has happened.4283

We still have X and Y on the outside but in this case, A to B went from left to right.4289

A to B goes right to left.4300

The last one that we are going to show is a deletion.4308

How can we take something out of the genome?4311

If we see here, we have X rightward arrow to B.4318

Nice stretch of DNA, A rightward arrow to Y.4325

What we can do is we can circle that up.4334

X to B, A to Y.4344

We can make our nice red cross again and we can draw it out.4358

Coming back down here, what do we have?4366

We are going to go X down to Y, that is the end of the DNA.4372

We are going to have a plus.4380

What about this one?4383

We can say this is A going to B and it is circular.4385

This is A going to B and it is circling around back to A.4397

This is almost back to up here right.4405

We have started with this again.4410

This is called deletion.4412

This is how you could invert insert something in.4420

You could insert a piece of DNA into a whole cell genome, think of viruses do this.4425

You can invert something, a piece of gene.4430

Or you can cut yourself out of the genome.4435

We think of our viruses that jump into the human gene and lay dormant for a while.4440

They have inserted in there.4447

When they want to be active again, they can cut themselves out, deletion, and become active again.4450

These are some examples of our site-specific recombination.4459

I hope you enjoyed this lesson and I hope to see you back.4462

Thank you very much for joining us at www.educator.com.4465