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DNA Mutations & Repairs

    Long, 5 examples, 5 practice questions

  • DNA damage is the alteration of the chemical structure of DNA.
  • Unrepaired damage can lead to a permanent change in the DNA sequence, known as a mutation.
  • DNA mutations are divided into two classes: spontaneous mutations and induced mutations.
  • DNA repair mechanisms commonly used by the cell are MMR, BER, NER, TLS, and recombinational repair.
  • DNA glycosylases are important enzymes involved in base-excision repair in both prokaryotes and eukaryotes.

DNA Mutations & Repairs

Which of the following is NOT an example of ionizing radiation?
  • Cosmic rays
  • Gamma rays
  • Ultraviolet rays
  • X-rays
Which of the following pathways is mainly responsible for repairing replication errors?
  • Mismatch repair
  • Translesion synthesis
  • Double-strand break repair
  • Base excision repair
Which of the following pathways is mainly responsible for repairing small, non-bulky lesions, using DNA glycosylases?
  • Mismatch repair
  • Translesion synthesis
  • Nucleotide excision repair
  • Base excision repair
Defects in which repair pathway may result in diseases such as Xerodoma Pigmentosum and Cockayne Syndrome?
  • Mismatch repair
  • Translesion synthesis
  • Nucleotide excision repair
  • Base excision repair
What is the least error-prone mechanism of double strand-break repair?
  • Non-homologous end-joining
  • Alternative end-joining
  • Homologous recombination
  • Translesion synthesis

*These practice questions are only helpful when you work on them offline on a piece of paper and then use the solution steps function to check your answer.


DNA Mutations & Repairs

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Lesson Overview 0:06
  • Damage vs. Mutation 0:40
    • DNA Damage-Alteration of the Chemical Structure of DNA
    • DNA Mutation-Permanent Change of the Nucleotide Sequence
    • Insertions or Deletions (INDELS)
  • Classes of DNA Mutations 1:50
    • Spontaneous Mutations
    • Induced Mutations
  • Spontaneous Mutations 3:21
    • Tautomerism
    • Depurination
    • Deamination
    • Slippage
  • Induced Mutations - Causes 6:17
    • Chemicals
    • Radiation
  • Example 1 8:30
  • DNA Mutations - Tobacco Smoke 9:59
    • Covalent Adduct Between DNA and Benzopyrene
    • Benzopyrene
  • DNA Mutations - UV Damage 12:16
    • Oxidative Damage from UVA
    • Thymidine Dimer
  • Example 2 13:33
  • DNA Mutations - Diseases 17:25
  • DNA Repair 18:28
    • Mismatch Repair
    • How to Recognize Which is the Error: Recognize Parental Strand
  • Example 3 26:54
  • DNA Repair 32:45
    • Damage Reversal
    • Base-Excision Repair (BER)
  • Example 4 36:09
  • DNA Repair 45:43
    • Nucleotide Excision Repair (NER)
    • Nucleotide Excision Repair (NER) - E.coli
    • Nucleotide Excision Repair (NER) - Eukaryotes
    • Global Genome NER
    • Transcription Coupled NER
  • Comparing MMR and NER 51:58
  • Translesion Synthesis (TLS) 54:40
    • Not Really a DNA Repair Process, More of a Damage Tolerance Mechanism
    • Allows Replication Past DNA Lesions by Polymerase Switching
    • Uses Low Fidelity Polymerases
    • Steps of TLS
  • DNA Repair 1:00:37
    • Recombinational Repair
    • Caused By Ionizing Radiation
    • Repaired By Three Mechanisms
    • Form Rarely But Catastrophic If Not Repaired
    • Non-homologous End Joining Does Not Require Homology To Repair the DSB
    • Alternative End Joining
    • Homologous Recombination
  • Example 5 1:09:37

Transcription: DNA Mutations & Repairs

Hello, and welcome back to

Today, we are going to talk about DNA mutations and repair.0003

As an overview, first thing we have to talk about is, what is the difference between DNA damage and the DNA mutation.0008

Once we understand that difference, we can talk about the various types of DNA mutations that we will come across.0016

When we come across mutations, hopefully, we do not even get that far and we can go through DNA repair.0024

We will talk about several types of DNA repair processes.0031

We will finish up getting ready to move on to the next unit.0035

First of all, what is the difference between DNA damage and DNA mutation.0042

DNA damage is just an alteration of the chemical structure of DNA.0046

That can be a break in the DNA backbone, we can have a loss of a nucleotide base.0051

Or we can have some sort of chemical alteration of that nucleotide.0056

DNA mutation is a permanent change of that nucleotide sequence.0061

The mutation will result from DNA damage that is not repaired properly.0067

It can also result from a replication, it can be exogenous or endogenous types of damages that are not repaired.0073

Or we can have what are called INDELS, it stands for insertions or deletions.0081

These are often a result of transposable genetic elements which we will call transposons.0099

Some other classes of the DNA mutations, the two main ones that we are going to see 0112

are either spontaneous mutations or induced mutations.0116

Our spontaneous mutations are our endogenous damage.0120

This does not result to molecular decay.0130

These are mutations that are due to error prone replication bypass, a naturally occurring DNA damage.0133

Usually, this is from error prone translation synthesis which is a type of DNA repair mechanism, 0140

that we will talk about later in this unit.0146

These errors are usually introduced during DNA repair.0149

We also have induced mutations, these are going to be exogenous damage.0152

This is going to be DNA damage caused by mutagens, whether that be chemical or radiation.0160

What we are eventually going to end up with, no matter which one we are looking at is, 0168

if we have damage and we do not have proper repair, we are going to end up with some sort of pathology.0173

Some sort of possible disease symptoms.0181

If we see here, a disease cell is when we have more damage than repair going on.0184

A healthy cell will be, when we have just as much repair going on, as we have damage.0191

It is almost like nothing ever happened.0198

Some of our spontaneous mutations that we can come across are tautomerism,0203

that is basically a cause of why certain bases will mispair with other bases.0209

Remember, our A’s should always double hydrogen bond to t’s.0216

Our G’s should always triple hydrogen bond with c’s.0223

Tautomerism is the repositioning of a hydrogen atom which alters the hydrogen bonding pattern,0228

resulting in incorrect based paring.0236

This could be an A maybe pairing with the G or C, or a G pairing with maybe an A or T, instead of that correct C.0238

Depurination is the loss of a purine base, forming what is called an apurine ExSite.0249

All that means is that, it is this site that does not have the purine base.0255

It does not have the nucleotide, the DNA backbone is still, the sugar, the deoxyribose is still there, 0261

we just do not have the proper base.0267

Another type is what is called deamination, that is a loss of an amino group.0271

Our amine groups are NH2 attached to the alcohol group.0277

We are losing amino group replacing that with a chytin groups.0285

Deamination of cytosine will lead to a uracil because cytosine and uracil only differ by that amine group.0288

If you deaminate adenine, you can end up with hypoxanthine.0299

If you deaminate your 5-methylcytosine, that will give you AT.0305

Let us just look at this one really quickly.0312

If we have a cytosine, cytosine should triple hydrogen bond our guanine up there.0315

If it is changed to a T, what are we going to do?0323

Now that T is going to hydrogen bond with an A, instead of the original G.0330

That can affect what type of mutations we see in future generations of DNA.0336

The last type of spontaneous mutation that we are going to talk about is called slippage or a slip strand mispairing.0344

What this is, is that we have denaturation of the new strand from our template during replication, 0351

that is followed by the renaturation in a different spot.0358

This can cause donations, w are going to talk a little bit about this slippage in a few slides, 0362

in which case I will draw out an example so that we can see what is actually going on here.0371

Some of our induced mutations and their causes.0379

Chemicals, chemicals are a type of what can induced mutations.0383

Some of the chemicals that can do that are, such things as base analogs, aquating agents,0390

DNA intercalating agents, DNA cross linkers, as well as oxidative damage.0397

Our alquating agents can mutate both replicating and non replicating DNA.0404

Base analogs can only mutate DNA that is undergoing replication, that is a difference for those.0416

Our turquating agents are molecules that get in between two strands of DNA.0425

I will show you an example of that when we talk about a tobacco smoke related mutation.0431

Cross linkers just covalent will cross link two pieces of DNA together.0439

Oxidative damage is something that we see all throughout.0444

Oxygen is just a molecule that can be good or bad, depending on what you are looking at.0447

We need oxygen to breath, excess oxygen especially reactive oxygen species can be very detrimental,0454

very reactive causing a lot of damage.0463

Another type of induced mutations or what can cause induced mutations is radiation.0466

We have non ionizing radiation, as well as ionizing radiation.0471

An example of IR would be X-rays, gamma rays.0477

IR produces really highly reactive free radicals that can break bonds in the DNA backbone.0488

If we break the DNA backbone that can be extremely detrimental, 0495

especially when trying to replicate new DNA or transcribe through portions of broken DNA.0500

Some examples of IR, as I mentioned before, we have our gamma rays, we also have our X-rays.0511

Remember, gamma rays are what is found in the Hulk, that turns Bruce Bane into Hulk.0519

X-rays, if you go in and get a scan trying to see through your body, looking at your bones as well, you encounter X-ray.0525

When you go flying...0531

Cosmic rays are what is in outer space, it is everywhere, it does not reach through the atmosphere very well.0538

They are quite abundant.0546

Some examples of our non-ionizing radiation are UV light, visible light, infrared, microwaves, and radiowaves.0550

Our ionizing radiation has a shorter wavelength and a higher energy.0560

Shorter wavelength, higher energy.0572

Whereas, our non-ionizing radiation has a longer wavelength, therefore, lower energy.0583

I said that I have mentioned what a DNA and intercalators.0600

What we have here, just down to the second bullet point is what we are looking out on the right side,0604

this is a covalent adduct between DNA and benzopyrene.0611

This is the major mutagen found in tobacco smoke.0617

What a DNA and intercalator is, benzopyrenes are DNA intercalators.0621

What a DNA intercalator is, it is something that binds in between the two DNA strands and pushes it apart.0626

If you see here the benzopyrene is right here.0635

It is affecting the DNA strand in its nice over all helical shape.0640

It is really pushing the two DNA strands apart and kind of the base interactions up.0647

Not only are we pushing outward, we are pushing the base stacking interactions down.0654

It is actually affecting, if we look at right here, these two nucleotides on opposite strands are not able to bind right there properly.0661

Or it may affect how many hydrogen bonds can be made.0673

Our benzopyrenes are categorized as pollutants, as well as carcinogens, something that can cause cancer.0678

They are naturally emitted by forest fires and volcanic eruptions.0686

They are also found in coal-tar. Cigarette smoke is I’m talking about right here.0692

This does not sound like something found in a product that we want to be putting inside of our bodies.0696

Another example of a DNA intercalator is ethidium bromide 0704

which is something that we use in a laboratory to help us visualize DNA.0708

However, it is mutagenic, it is carcinogenic, it can cause cancer because it will get in between pieces of DNA and cause alterations.0713

This can affect the DNA in future generations, if this causes a problem in the proper replication or transcription of the DNA.0724

One DNA mutation that I want to talk about is caused by UV light.0739

Remember, UV light is a type of non-ionizing radiation.0744

Oxidative damage occurs from UVA exposure.0750

One of the many things we have, occurring from this is what is called a thymidine dimer.0755

What that is, it is a covalent bond between adjacent thymines on DNA, 0761

on the same strand of DNA which forms a cyclobutane ring within the t’s, 0768

which does not allow them to properly base pair with A’s on the opposite strands.0777

This would be normal, and this right here would be our cyclobutane.0783

We have T's binding together here, instead of across the way to the a’s.0791

This is highly mutagenic and this can cause big time problems because you cannot replicate or transcribe through these places.0799

We have to repair them.0809

Let us talk about this real quickly.0815

Our UVB rays, they are shorter wave length which means they usually have higher energy.0822

Shorter wave length, higher energy.0829

They usually do not penetrate pass the epidermis, it is the outer stimulator.0832

UVB rays are the main cause of sunburns and skin cancers.0838

UVB rays account for about 5% of the total UV light that reaches earth.0844

UVA rays are longer and therefore less, lower energy.0854

They will penetrate through the epidermis down deep into the dermis, below that top layer.0863

UVA ray exposure may lead to premature skin aging, wrinkling, and immune suppression.0872

UVA rays are about 95% of the total UV light that reaches earth.0880

We also have UVC rays.0892

UVA is the dominant tanning ray.0895

UVB is going to sunburn, UVA more likely to tan you.0910

Now that we know what UVA and UVB are, let us talk about what we want to know regarding this.0922

We want to know how is this affect us when we got into the sun and how can we protect our self?0929

We all know about sun block and we have probably all seen the SPF factor.0936

The SPF 10, 15, 30, 50, SPF 90.0942

What is that actually mean?0948

SPF is what is called sun protection factor.0950

What that tells us is, it says how long it will take UVB rays to redden treated skin compared with untreated skin.0955

For example, if we take a sun block that says it is SPF 15.0966

What that means is that, if we put it on, say our left arm and not on our right arm, it will take our left arm, 0972

the treated skin 15 times longer to get to the same redness ,as this arm.0981

If I put this arm out for an hour in the sun, I will get some sort of redness.0992

Treated with SPF 15, this arm would take 15 hours of the same sun exposure to get to the same redness level.1001

This is a way that we can protect ourselves from these harmful rays.1009

Remember, our UVB not only the main cause of sunburns but also main cause of cancers.1013

Just as another side, those fake tanning booths, they are not much safer than the sun.1021

You still would need to use your SPF because actually fake tanning increases your risk for skin cancer by at least around 2 fold.1029

If we do not repair our DNA, we will lead to mutations.1048

Here is a list of many separate mutations that can be found from originally having a mutation in DNA.1054

And then right here, we are showing the codon table so that mutation with DNA gets transferred to the RNA.1062

And that mutation in the RNA, from DNA to RNA’s transcription, and the mutation transcription,1070

RNA to protein translation will give us a problem in a functional protein or1078

maybe a problem making up protein nonfunctional anymore.1083

What we are saying here is that DNA errors not repaired make DNA mutations.1088

DNA mutations can lead to disease, we want to be able to figure out a way to stop that.1096

Let us look at the possible repair mechanisms.1104

We are going to talk about all these today and that we have base-excision repair, nucleotide excision repair.1110

Recombination repair, homologous recombination, non homologous end joining, 1121

as well as one that is not listed on here, which are called alternative end joining.1126

We will start with mismatch repair and we will briefly mention direct reversal.1130

That is not so much, our repair making, as it is exactly what it says.1135

It is the direct reversal, let us say before repair mechanism would have to come in.1141

A mismatch repair says up here, mainly we are fixing replication errors.1147

Mismatch repairs, we are going to fix our errors in replication.1156

The normal error rate of replication is 〖10〗^(-5), that is 1 every 100,000, 1/100,000 bases, that is great.1160

But remember, we have over 3 billion bases in the haploid genome.1172

We need to get better than that, or else there are too many errors that can lead to mutations.1176

Maybe, it can lead to apoptosis or death of those cells.1180

If you have too many cells dying, as the organism will eventually die.1184

Proofreading mechanisms increase that to 1 in 10,000,000, that is great but still not good enough.1189

MMR, mismatch repair, can bring this to 1 × 〖10〗^(-10).1212

That is 1 in 10 billion bases, we only have a 3 billion base pair genome.1228

This is saying, by what it is, we should not have any errors during entire replication cycle, that is a great thing to see.1233

That is what we want.1243

MMR has to repair our mismatches, otherwise, it become mutations.1245

Mutations can lead to disease.1253

How does MMR repair our mismatches?1255

First thing, if we look down here, on the left side, this is how eukaryotes utilize mismatch replication.1259

On the right is how E coli utilizes mismatch replication or mismatch repair, MMR.1268

And then, we have the majority of bacteria is what you see in the middle.1276

I’m going to talk about the eukaryotic, since we are eukaryotes, as humans.1281

First thing, you have to recognize the mismatch.1288

Over here, we see a GT, that is not a proper mismatch.1291

We should have either an AT or a GC.1295

The mismatch is recognized by MutS α, that is a protein.1301

Then, we have an incision in the DNA backbone, that is done by MutL α.1308

Next, we will move the stretch of the DNA including that mismatch and that is done by XO1, it is a nuclease.1314

As well as RPA being involved, remember that is going to have bind to the single stranded DNA.1325

Once we have removed that, we can synthesize new DNA.1332

We have DNA polymerase Δ coming in, synthesizing new DNA.1336

Finally, DNA ligase, ligating the gap or the nick in the DNA backbone.1339

It is the same thing happening in bacteria, as well as E coli.1346

It is just different protein names.1354

An important thing to see is how do we recognize a mismatch in the first place?1359

How we see that mismatch is due to the fact that this is somewhat bulky.1366

This mismatch is bulky and that is the how this original protein, MutS α, 1372

if we are talking about eukaryotes, finds that mismatch.1379

Just as important of a question is, how can we tell which of the two strands has the error?1384

Which one is actually what it is supposed to be?1394

Is the G the error or is it the T the error?1397

How you figure out which one has the errors, you have to recognize 1405

which one is the parental strand vs. which one is the newly synthesize nascent strand. 1409

The parental strand will have what the proper sequence should be.1415

The error will be in the newly synthesized strand.1418

In E coli, how you recognize a parental strand is that MutH will bind to methylated sites on the DNA.1422

It will nick the non-methylated strand.1433

In E coli, they find these sites that are called GATC.1435

This adenine will have a methyl group added to it.1442

This occurs in E coli as a defense mechanism, so that any non-methylated DNA that is in the cell, it sees as foreign, it will cut it up.1449

Let us say a virus is trying to kill the bacteria, the virus latches on, expels its DNA in their.1464

The E coli can utilize a defense mechanism that breaks up any DNA that is non-methylated.1474

You cannot methylate DNA right away after synthesis.1482

There is a short period of time where the newly synthesized DNA is unmethylated.1486

That is how E coli can recognize the parental strand vs. the newly synthesized strand.1491

Once it is recognized, we have a wJ which is a 5 prime to 3 prime exonuclease 1497

or EXO1 which is a 3 prime to 5 prime nuclease.1503

It will bind and remove that stretch of DNA, including the mismatch from only the non-methylated strand.1507

It will not do this on the methylated strand.1516

This will only be removed from the newly synthesized strand leaving the correct parental strand sequence there.1520

When you go to the resynthesis process, you will hopefully this time synthesize across the proper parental strand, 1528

giving you the correct AT base pair, instead of what it was shown was a GT.1538

Eukaryotes and prokaryotes do not methylate their strands.1549

This one is a little bit harder to think about.1554

Our parental strands are unbroken during replication, we know that.1559

Our nascent lagging strand, as we have already talked about, has nicks because of the Okazaki fragments.1565

The polarity of how DNA polymerase synthesize 5 prime to 3 prime, leaves us with a lot of nicks.1572

That is how eukaryotes and a lot of prokaryotes, can recognize which strand is parental vs. which strand is newly synthesized nascent.1578

We have XO to 3 prime to 5 prime exonuclease, binding and removing that stretch of DNA,1592

including the mismatch from the nascent strand only.1597

And then, we have our DNA poly-Δ coming back in, resynthesizing, hopefully, this time the correct nucleotide.1601

We had DNA ligase sealing the gap.1609

I said earlier that we talked about slippage and I would draw this out so we can understand it a little more.1617

Mismatch repair can also repair loops in the nascent strand, due to what is called slippage.1625

If we have a piece of DNA, this would be a GTC, GTC, GTC. 1632

If we have repeat sequences, this can be problematic during replication1661

because what can happen is that we can have, what I said is called slippage.1669

During slippage, we can have some DNA alteration, to where we have it coming off of the proper sequent.1681

What we have, we can draw this like this so it looks easier.1699

This one is actually binding to this.1719

What we have is, if this happens, if we slip this loop upward but we are still binding properly here, right,1732

this is all hydrogen bond, we end up with an increase in our DNA.1744

We end up with what is called excess repeats.1750

If we write this over here, this is going to be an expansion.1756

In this case, it is a triplet expansion because there are 3 bases.1762

These are going to end up being added in, when they were not before.1767

Let us draw this again, we have CAG.1773

These have gotten added.1779

What is going to end up happening is that, we are going to end up having to form this in this excess, GTC, GTC, GTC.1803

What would happen is we can have, this is our mutant DNA, this has all been added.1818

This is problematic because as a triplet repeat, specially when we are talking about this one right here,1830

this is what we call a polyglutamine repeat.1836

The reason being that the CAG, this will code for glutamine, when we are talking about turning it into mRNA, 1849

and then finally into a protein, this will code for glutamine.1863

There are several diseases that are associated with polyglutamine expansion.1869

A couple of those are Huntington's disease, as well as spinocerebellar ataxia, 8 different types of that.1875

If we are talking about just triplet expansions not necessarily the polyglutamate ones,1910

we also have diseases such as fragile X syndrome, as well.1914

These can be extremely detrimental.1920

What we have here is, the more repeats that we see, the more likely we are to go through slippage, 1924

the next time you go through DNA replication.1931

We have mismatch repair, hopefully being able to catch this, remove these.1934

Hopefully, we can remove these so that we do not have the expansion and 1944

we just can go back to our regional DNA sequence there, so that we do not have the expansion and we do not lead to disease.1949

MMR plays a huge part in that.1960

I told you we would very briefly mention damage reversal.1967

What damage reversal is, it is exactly what it says, you are reversing the damages 1973

before you have to go to a true DNA repair mechanism like mismatch repair or the other ones that we are going to talk later.1978

An example of damage reversal is the direct reversal of pyrimidine dimmers.1986

Remember, our pyrimidines are c’s, U’s and t’s.1994

If we are talking about DNA, hopefully, we do not have any uracil in our DNA.1999

If we are talking about DNA, hopefully, we are just saying cytosine and thymine.2005

Our pyrimidine dimmers, often as we talked about before, caused by the UVA light, the thymidine dimmers.2010

Direct reversal is catalyzed by, in this specific case, DNA photolyase.2018

DNA photolyase is an enzyme that would use energy from visible light to break the cycle of UV ray formed between thymidines.2024

If we look here, here is our DNA, our thymidines are bonded together instead to the adenines across.2035

If we can get photolyase to come in there, it can break that bond and2048

help make the proper bonds across the way between our adenines and thymines.2055

That is an example of damage reversal.2070

Onto other type of repair, this is a very important one, all of them are very important ones.2074

This is one that we are going to use for a small and non-bulk lesions, however, we see these in descent amount.2082

Base-excision repair, otherwise known as BER, uses specific DNA glycosylases2092

to remove abnormal bases that are due to oxidation and alkylation, or deamination.2101

These types of mutations are often caused by IR, ionized radiation, aquated agent, oxygen radicals, or spontaneous hydrolysis.2109

Hydrolysis is just the breaking of something.2120

We see a damage base, the glycosylase comes in there, recognizes it.2126

Recruit a nuclei and endonuclease to cut that piece out.2133

And then, it will just resynthesize the correct base and re-ligate that gap.2138

We have DNA poly-β being involved in short patch repair, if we look on the right side.2144

We have DNA poly-Δ and ε involved in long patch repair for DNA synthesis.2152

All that means is the length of the repair process.2162

Few bases, several bases.2165

Here is an example, if we have the spontaneous deamination of cytosine, let us draw this out.2171

Here is the DNA sequence, AT, CG, AC, TG.2180

That gives you TA, GC, TG, AC.2192

If we have spontaneously deamination, that is when we have our main group coming off.2203

What that leads us to is ATC, CGA, CTG, and then, it is TAG.2236

Let us say this C was the one that got deaminated.2263

Remember, I told you before that C can be deaminated to a U.2268

This is TG, AC.2286

Once we see that spontaneous deamination, we have what is called uracil glycosylase coming in and2300

cutting this base from the backbone, leaving what is called the A basic site.2307

If we continue on this way, we have uracil glycosylase.2314

AT, CG, AC, TG, we have TAG, TGA.2351

Right here, we no longer have the base because it is been removed by uracil glycosylase.2369

It is cutting at the place of where the uracil is attached to the deoxyribose.2378

It is hydrolyzing that one.2387

The next thing that will happen is that we have AP endonuclease making the phosphodiester backbone which is 5 prime to the A basic site.2390


We got to nick that backbone, 5 prime to that A basic site.2472

The basic site just means it is a site without any base.2476

Abasic means it can be a purine, it could be a pyrimidine.2481

Here is in abasic site right here, that we are looking at.2484

Here we have the nicking of backbone due to the action of AP endonuclease.2488

The next step, we have deoxyribose phosphate lyase removing the deoxyribose from the backbone.2497

Let us see this, I will write out what we were starting with.2506

We have ATC, GAC, TGT, AGT, GAC.2512

This is just right after AP endonuclease has worked.2540

We have deoxyribose phosphate lyase.2551

What we have here is, we are going to remove the deoxyribose from the backbone.2573

What we have is 5 prime AT, CG, AC, TG.2583

We have TAGTGAC.2598

AP endonuclease over here just made a nick but it did not remove the actual deoxyribose.2610

That is where deoxyribose phosphatelise comes in, it removes the entire thing.2617

We have a full break in that backbone, right there.2624

We are missing the sugar.2627

The final thing happens is we have the DNA polymerase and DNA ligase coming in.2631

Remember, add a new deoxy CTP, removing a pyrophosphate, that enters as a monophosphate. 2676

We end up with the repaired DNA, ATCGACTGTAG.2686

The C, as well as the new deoxyribose has been added and we are back to normal, what we had.2715

This is the full repair using base-excision repair.2723

As I said, DNA polymerase and DNA ligase bring in the correct cytosine nucleotide and they seal the nick in that phosphate backbone.2733

It is good as new, like it never happened.2741

Another type of repair, just switch a little, is nucleotide excision repair, otherwise known as NER.2745

NER recognizes a wide range of our DNA damage, including our damaged bases, 2756

the pyrimidine dimmers that we have already talked about, as well as bulky adduct, those are just big crosslink molecules.2761

Defects in NER can result in disease.2769

A couple of diseases to mention is XP xeroderma pigmentosum, that often presents with photosensitivity.2776

You are very sensitive to light getting into skin regions and cancer.2786

Down here is a patient with XP, you can see that they have a lot of skin pigmentation.2795

This is due to the fact that we are accumulating these problems from UV light, these damage bases dimmers, all these things.2805

We need light all the time, we need to figure out a way to repair them.2815

When we have a problem in a main repair process, that builds up over time.2820

Actually, people with XP cannot be in sunlight for very long, by any means.2825

When they do go outside, they have to go out fully clothed, almost like a bee keeper’s uniform.2831

They have a hat with a face shield, their body should be covered 2838

so that you do not end up with the accumulation of all this mutation.2845

Another defect of NER can produce another disease called Cockayne's syndrome, that also presents with photosensitivity.2850

As well as growth failure in their gene generation.2864

Let us talk about nucleotides excision repair.2874

We will talk about it being in E coli first because the mammalian version is, while it is very similar,2876

it is much more complex and has 25 or more proteins involved.2883

Same concept, just a lot more complicated.2887

Let us talk about E coli.2890

First thing that happen is, UVRA and UVRB proteins scan the DNA for distortions.2893

This is number one down here.2899

UVRB will melt open the helix, next.2903

UVRC will cleave on both sides of the lesion, about 12 to 13 base pairs, this is 3.2910

Next, UVRD helicase will remove the lesion, this is 4.2921

DNA polymerase 1 will fill in the gap.2929

DNA ligase will seal that nick, number 5.2935

It is a fairly simple concept but a very important part, very important for the proper movements of the genome.2949

I have it listed later, just so you know, UVRA and UVRB, in humans, the protein that performs the same function is XPC.2961

UVRB, the human analog would be XPA, XPD.2973

UVRC that is similar to XPG and ERCC1 XPF complex.2987

UVRD, it does not really have as going to be an analog.3002

Instead of a 12 to 13 base paired gap in bacteria, in humans or eukaryotes, we have about a 24 to 32 base pair lesion.3009

As I said, many more proteins involved.3025

There are two different types of NER that we can accomplish or complete in eukaryotes.3030

First is global genome nucleotide excision repair.3037

The second one is transcription coupled NER.3041

The difference is that global genome NER can repair damage in both the transcribed, 3045

as well as non-transcribed DNA strands in both active and inactive genes throughout the genome.3052

Transcription coupled NER can only be performed on actively transcribed genes only.3060

Transcription coupled NER, TCNER, rescues a stalled RNA polymerase at whatever this lesion is.3074

What we have is transcription factor 2H, TF2H participating in general transcription, 3083

as well as transcription coupled NER.3090

It is already there, it participates via its XPA and XPD units, the helicase.3093

This is important to help get RNA polymerase 2, to remove this lesion and continue on to transcription.3101

Otherwise, we can have problems and eventually lead to the cell undergoing apoptosis.3111

I talked about apoptosis, remember, apoptosis is the programmed killing of cells.3120

We talk about that being a good thing, sometimes, because if we do not go through apoptosis, 3128

you can end up having unchecked cell proliferation which we know leading to cancer.3133

Let us compare MMR and NER.3141

It does not matter which one we are looking at, they all go through a similar series of steps.3154

First things first, there is a surveillance of the DNA damage lesion.3161

You either recognize that there is a problem then, you induce a cell cycle checkpoint.3168

You do not want the cell to continue on like it is normal, you need to repair that lesion first.3174

You activate those repair networks.3180

You will go through DNA repair and you will keep this cell cycle in an arrest.3183

You do not let it go on any further.3190

You can go one of two ways at that point, either you have figured out the repair cannot be done.3194

You go and finish with apoptosis, you kill the cell.3203

It is beyond repair, we are just going to cell and we will start over.3210

Or we go through and we proofread, we repair, we do everything.3214

We think that we are on the right track.3219

We are going down this way.3222

From there, we have two options once again.3225

Everything is okay, we go on to the next process and whatever it is supposed to do in the cell.3229

From replication, we can go S phase then we can start going through mitosis.3237

If we still see that there is a problem, we can have a part point where we can go through apoptosis.3242

Too much has happened, we did not fix it correctly, we are going to kill that cell, we will start fresh.3249

Both of these pathways are very similar in that effect of, we look for the damage, we induces cell cycle checkpoints, we stall it.3257

We repair and we check to see if we actually repaired it properly, in which case we can continue on.3269

Or if we did not repair it properly, we kill the cell off.3274

One of the last things that I’m going to talk about is what is called translation synthesis.3284

Translation synthesis really is not a repair process.3291

I know that this is in the DNA repair section, we need to talk about it.3296

Transition synthesis is also abbreviated TLS.3301

It is not a repair process, it is more of the damage tolerance mechanism.3305

This can be induced by our cell cycle checkpoints, if we are talking about eukaryotes or bacteria.3311

SOS response is basically the same thing.3317

This TLS allows replication pass these DNA lesions by polymerase switching.3321

You may find, let us say an example, AT.3329

It is usually going to be little more involved than that but it should be an AT but we see an AG.3334

Maybe we see a bunch of these.3343

That is problematic, it stops synthesis replication.3346

We cannot go through their, we cannot figure out a way to get pass it.3352

I will give you a better one.3356

Let us say that the pyrimidine dimers, these are attached to each other instead of across the Y to A.3363

We cannot get through there, instead of trying to synthesize, we just throw in another polymerase 3369

that does not care what the correct nucleotide should be , it just throws stuff in to get past the gap.3376

How it does this is, it goes to polymerase switching and it uses low fidelity or very error prone polymerases.3387

These polymerases will incorporate bases that likely do not match the proper base paring to the template strand.3395

These error prone polymerases are usually from the Y family of polymerases.3404

If we are talk about E coli, we have polymerase 4 and 5.3408

In eukaryotes, we poly-aeta, poly-aota, poly-kappa.3414

The cool thing about poly-aeta is that it automatically inserts two adenines across from a thymidine dimer.3420

When we see this thymidine dimer and we need to synthesize this new strand down here, 3426

we cannot utilize this TT as a nice template.3438

What it does is, this poly-aeta says, I see a TT, whatever, I’m just throwing two A’s in there and continue on with my synthesis.3447

Luckily, we have a failsafe for that, that we can get through this process.3457

It is error prone, it does necessarily know that there are two t’s.3464

The steps of TLS, first the lesions encountered and we start a replication fork.3470

Secondly, we have the PCNA, the sliding clamp on our replicative polymerase, gets ubiquitinated.3475

Ubiquitin is just a very small protein, it can be added to our sliding clamp.3483

That will cause the sliding clamp to release from DNA, the sliding clamp and the replicative polymerase.3490

We can switch to a translesion synthesis polymerase that bypasses the lesion, regardless of what that sequence is.3498

It could be TTA, CGT, TCA.3505

We are trying to synthesize this way.3521

Let us say we find the lesion being around this area.3528

This is fine, we have a T, we have a G, we have an A.3533

We run into a lesion, the replicative polymerase falls off.3543

Let us it ε just for sake.3553

We get our trans-lesion synthesis polymerase coming on, poly-kappa.3557

Poly-kappa comes on, it is going to start polymerizing.3568

Maybe it gets a couple right, maybe it just gets it once, it does not care.3574

It is just trying to get through that lesion.3581

We will say the lesion stops here, at which case we have the TLS coming off.3583

We have our replicative polymerase coming back on and synthesizing with high fidelity again.3592

This right here, this area might not exactly match properly.3603

It is more useful for the cell to be able to have a few mutations and get through,3612

to be able to finish all replication than for the cell to go through apoptosis.3619

Yes, this could end up causing cancer but it might also end up leading to evolution, 3624

as can be seen throughout many generations, so far.3631

The last type of repair I’m going to talk about is recombination repair.3639

This is going to give you a nice overview and preview for what we are going to do in the next unit.3644

Recombinational repair repairs our double strand breaks.3654

These double strand breaks are caused by ionizing radiation.3659

If you remember, ionizing radiation are cosmic rays, gamma rays, X-rays.3663

As I have mentioned before, you can get X-rays from imaging, from radiation therapy, from flying, all kinds of ways.3669

We have our double strand breaks being repaired via three main mechanisms.3677

We have NHEJ, non-homologous end joining.3681

We have alternative end joining which we call ALTEJ.3684

We have homologous recombination which we can call HR.3689

We are going to talk about these, specially homologous recombination, we are going to talk about in depth, next time.3694

Double strand breaks form pretty rarely, but they can be catastrophic to the cell, if they are not repaired.3706

On average, we have about 10 double strand breaks per cell per day, occurring from replication alone.3714

Not to mention all the other things, our X-rays, the exogenous stuff.3725

If we do not repair these double strand breaks, we can lose pieces of chromosomes.3732

That can be extremely detrimental and hopefully that could lead to, if unrepaired, apoptosis.3737

You will have cell death. 3744

If not repaired and does not go to apoptosis, you can likely form some sort of cancers.3746

The mechanisms of double strand break repair are the three that I mentioned before.3754

Non-homologous end joining is our fastest one.3759

It takes a little less than an hour but it is really error prone.3764

When is it usually occurring, in the G1 phase or in the G0 phase, the senescent cells, the non dividing cells.3768

We have ALTEJ, alternative end joining.3776

That is used very rarely, it is really slow, about a few hours long.3784

It is also error prone.3791

We have the preferred mechanism, homologous recombination.3794

It is the slowest, it is about 8 to 10 hours but it is fairly error free.3800

This is usually occurring in S phase or G2.3805

This is in S phase, when we are having a lot of, hopefully not mutations but errors, DNA damage.3810

Non-homologous end joining, this does not require homology to repair the double strand break.3825

What it does, we have a protein complex, heterodimer, Q7080, 3831

binding the double stranded ends and recruiting a kinase called DNAPKCS.3838

DNA protein kinase, CS catalytic subunit.3845

We then have artemis in XO1 endonuclease, removing mismatch nucleotides via resection, cutting away the ends.3852

We then finally have synthesis by poly-μ and poly-λ.3860

We ligate ends via DNA ligase 4.3864

This will result in point mutations and small deletions.3868

The double strand break is repaired.3873

What the NHEJ will look like just very simply.3876

We have our Q7080 binding then you recruit the kinase, artemis.3888

It has removed that piece then we can get proper synthesis.3899

ALTEJ, alternative end joining, uses microhomology to rejoin the two ends of the double strand break.3909

What that means, microhomology, is it finds some sort of match, a minimum of 4 base pairs long, between the two strands.3916

How that would look?3926

Let us just write that in there.3958

They find this, there is a double strand break, which is seen here.3978

You search down each individual piece, to where you can find a microhomology that at least 4 bases long.3986

We reset the ends using the mRNA complex and CTRP, and we find that microhomology.3998

Just a little bit of base pairs that would match each other on opposite strands.4007

Then you re-anneal via that microhomology.4012

What we have is, we have flaps that are created.4018

What you can do is, you have an endonuclease coming in, breaking off, cutting those flaps, then, you re-ligate across.4023

What happens is, each one of those flaps that you have cut off, that contain a bunch of DNA sequent.4038

That can result enlarge the lesions, which is why alternative end joining is used as a last resort.4045

Because, you have lost all this sequence, that can be a bad thing.4053

Finally, we have recombinational repair.4061

We have homologous recombination HR, which repairs double stranded breaks 4064

using long homologous sequences, as the name would call out.4070

They use homologous sequences as templates for synthesis.4077

What is very indicative of HR are these strand invasion event and the holliday junction formation.4080

The main steps of recombinational repair via homologous combination is resection, same thing.4094

We have resection occurring here, we are cutting away the DNA.4102

This is now missing.4106

We have strand invasion which is occurring right here, where this strand is going into the other DNA duplex of homologous chromosome.4111

Here is the strand invading, then, you have holliday junction formation.4121

A holliday junction is this right here, it is also this.4127

We can resolve this either by double strand break repair mechanism or by synthesis dependent strand, either one of these.4134

We are going to go into more detail, what each of this one looks like in the next lecture.4145

But quickly, double strand break repair, we have the formation of our double holliday junctions, we have branch migration.4150

We can have either non-crossover or crossover products, whereas synthesis dependent strand annealing, 4159

we just have strand displacement.4167

We do not have any type of cleavage and we usually results in no cross over product.4170

I will leave you with this right here.4179

This is a table I have put together to help you with each of the repair processes.4181

What type of damage that they will focus on and the enzymes that involved.4187

Mismatch repair is going to focus on replication errors.4192

Those are going to be our MutS, MutL, MutH.4200

Humans are MSH, MOH, PMS, these are ones that are listed on the previous slide before.4205

We have base-excision repair, mismatch repairs MMR.4216

Base-excision repair, BER, that is usually used to fix damage caused from ionizing radiation,4222

oxygen radicals, alcalated agent, or spontaneous hydrolysis.4234

The enzymes involved are DNA glycosylases and AP endonuclease.4238

This is for both prokaryotes and eukaryotes.4242

We then go to nucleotide excision repair, NER.4247

We can use this to fix pyrimidine dimers, bulky adducts from non-IR, our UV light, our thymidine dimers.4252

This is what a UVR complex is in E coli come in.4263

Or the humans, we have the XP complexes, as well as about 25 other proteins.4267

Trans-lesion synthesis, TLS, we do this when we come across an unrepaired pyrimidine dimer,4275

when we come across a site that does not have a base.4283

Or if we have a bunch of bulky adducts that have gone through one of these4287

MMR, BER, NER processes and have not been fixed properly.4291

E coli, whether it is E coli or humans, the Y family polymerases come in.4297

These are the error prone polymerases that synthesize through these gaps.4304

Remember, this is more of a damage tolerance mechanism than a repair process.4309

The Y family polymerase is found in E coli, our polymerase 4 and 5.4314

The Y family ones taking part in humans or eukaryotes,4319

is poly-aeta, poly-aota, and poly-kappa.4324

We have our recombinational repair which is taking care of our double strand breaks.4329

This is going to include, our recombinational repair is going to be NHEJ, ALTEJ, and homologous recombination HR.4336

In E coli, like what we see, complex is involved in that.4353

In humans, Q7080 heterodimer, as well as DNA PKCS and artemis are involved in human side of that.4358

We will go over recombinational repair, and specifically homologous recombination, in the next unit.4367

That is the end of today's unit, thank you for joining us at

I hope to see you again soon.4387