DNA Instability, Endogenous Mutagenesis, and DNA Repair
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00:00
NobeliumChemische ForschungAusgangsgesteinGangart <Erzlagerstätte>MolekülAdvanced glycosylation end productsIdiotypDoppelhelixDNS-SequenzChemische ForschungDNSErdrutschFreies ElektronVorlesung/Konferenz
00:54
DNSAdeninChemische StrukturBiochemikerinDNS-DoppelhelixTopizitätBasenpaarungDuplikationChromstahlMakromolekülMolekülErdrutschChemische ForschungEukaryontische ZelleBesprechung/Interview
02:19
GuaninCytosinDNSAdeninRadioaktiver StoffDNSZusatzstoffNeutralisation <Chemie>Volumenhafter FehlerPufferlösungZellwachstumIsotopenmarkierungChemische StrukturNucleinbasenDoppelhelixAuftauenNucleotideSubstrat <Boden>CytosinKörpertemperaturGuaninPeriodateWässrige LösungAktives ZentrumAbschreckenGermaneGezeitenküsteMemory-EffektStoffwechselPeroxyacetylnitratKohlenstofffaserVorlesung/Konferenz
04:04
AdenomAdeninDNSGuaninCytosinMedroxyprogesteronTellerseparatorIsotopenmarkierungIonenbindungEukaryontische ZelleVerbundwerkstoffKohlenhydratchemieProteineKörpertemperaturVerrottungChemische ForschungAngelicasäureNucleinbasenDNSRadioindikator
05:47
DNSNobeliumSimulation <Medizin>FleischerDNS-SequenzRückstandNucleinbasenEnzymKohlenhydratchemieIonenbindungGuanin
06:12
DNSGuaninAdeninCytosinNobeliumNucleinbasenEnzymAktives ZentrumKohlenhydratchemieRückstandGuaninDoppelhelix
06:39
GuaninCytosinDNSRadioaktiver StoffDoppelhelixAzokupplungOktanzahl
07:09
NucleotideDNSSelbstzündungEukaryontische ZelleLäsionNobeliumGuaninEukaryontische ZelleArzneimittelReaktivitätAlkylierungOktanzahlNucleinbasenDNSMolekülDNS-SchädigungEnzymDNS-SequenzReaktionsmechanismusLäsionDesaminierungCytosinPyrimidinOxidschichtDNS-DoppelhelixStrahlenschadenAktives ZentrumRadonReplikationAdvanced glycosylation end productsSonnenschutzmittelPegel <Hydrologie>HydrolysatVorlesung/Konferenz
09:56
NobeliumDNSHydrolysatFleischerChemische StrukturDNSGuaninEukaryontische ZelleCytosinAktives ZentrumKernproteineKatalaseVorlesung/Konferenz
10:34
DNSNobeliumFilmscharnierNucleinbasenDNSNucleotideDNS-abhängige-DNS-PolymerasenLyasenEukaryontische ZelleBeta-FaltblattGap junctionFunktionelle GruppeNeotenieVorlesung/Konferenz
11:30
DNSNucleinbasenProteineSauerrahmEnzymEnzymMannoseSystemische Therapie <Pharmakologie>BaseChemischer ProzessErdrutschNucleinbasenComputeranimationVorlesung/Konferenz
12:01
NobeliumNucleinbasenProteineMagmaEnzymDNSCytosinGangart <Erzlagerstätte>RadonVerletzungNucleinbasenDNSEnzymIonenbindungUracilRNSGlykosylierungFleischerCrackComputeranimation
12:34
NobeliumProteineFilmscharnierOligonucleotideKernproteineEnzymGangart <Erzlagerstätte>Kettenlänge <Makromolekül>IsotopenmarkierungNucleotideStoffwechselwegUracilDNS-DoppelhelixAcetonElektronische ZigaretteNucleinbasenAktives ZentrumDNS-abhängige-DNS-PolymerasenAlkalitätSystemische Therapie <Pharmakologie>StrahlenschadenAlkoholische LösungDNSBaseZuchtzielWursthülleGezeitenküsteStoffgesetzACEVorlesung/Konferenz
14:19
DNSStrahlenschadenDauerwelleImmunglobulineAntikörperDNS-SchädigungMitochondriale DNSAntigenitätGenDNSKettenlänge <Makromolekül>StrahlenschadenEukaryotenHerzfrequenzvariabilitätChemischer ProzessReaktionsmechanismusACEBleierzBukett <Wein>Computeranimation
15:46
NobeliumStrahlenschadenDNSUracilDesaminierungAktives ZentrumAntikörperAktivität <Konzentration>DNS-SequenzDNSBukett <Wein>Chemischer ProzessStratotypGen
16:20
DNSStrahlenschadenImmunglobulineDauerwelleOrlistatCytosinRedoxreaktionDNSStrahlenschadenGenHerzfrequenzvariabilitätOxidschichtStoffwechselHydrolysatWasserfallSetzen <Verfahrenstechnik>CobaltoxideIdiotypComputeranimation
17:42
NobeliumDNSAdeninCytosinRedoxreaktionGuaninFilmscharnierAmine <primär->DNSThermoformenStrahlenschadenReaktionsmechanismusBleioxid <Blei(II)-oxid>Chemische StrukturRückstandGuaninNucleinbasenReplikationIdiotyp
18:14
DNSGuaninCytosinRedoxreaktionAdeninDiatomics-in-molecules-MethodeNobeliumBiskalcitratumReplikationDNSIonenbindungGlykosidasenNucleinbasenEnzymKohlenhydratchemieAktives Zentrum
18:54
DNSRedoxreaktionGuaninAdeninCytosinNobeliumAdenosylmethioninWasserMethylgruppeEukaryontische ZelleReaktivitätChemische ReaktionMolekülDNSAktives ZentrumFunktionelle GruppeCobaltoxide
19:47
NobeliumBiskalcitratumDNSAktives ZentrumProteineDNSChemischer ProzessRNSAlkylierungReaktionsmechanismusChromosomBleierzOrganische ChemieAzokupplungSense
20:39
DNSGuaninCytosinAlkylierungAdeninNucleinbasenMethylierungReaktionsmechanismusBiskalcitratumNobeliumNitrosamineAzokupplungAktives ZentrumMethylgruppeDNSNucleinbasenEukaryontische ZelleReplikationEnzymIdiotypSiedenMolekülDoppelhelixInhibitorSalzsäureIonenbindungPharmazieRadikalfängerSystemische Therapie <Pharmakologie>ProteineFunktionelle GruppeGasphaseComputeranimation
22:33
ReaktionsmechanismusMethylierungDNSNucleinbasenNobeliumCysteinRadikalfängerEukaryontische ZelleProteineThermoformenKüstengebietStromschnelleComputeranimation
23:16
NobeliumDNSMethylierungReaktionsmechanismusKonkretionTiermodellVerletzungLäsionProteineReplikationGesundheitsstörungCytosinMethylgruppeDNS-SchädigungThermoformenAktives Zentrum
23:48
DNSReaktionsmechanismusMethylierungNucleinbasenNobeliumBiskalcitratumTiermodellProteineHomologisierungEukaryontische ZelleAzokupplungGenklonierungGenComputeranimation
24:18
DNSReaktionsmechanismusMethylierungNucleinbasenNobeliumEnzymMetalloenzymChemische VerbindungenSonnenschutzmittelMolekülChemische ReaktionMischanlageAlphaspektroskopieComputeranimationVorlesung/Konferenz
24:50
BlauschimmelkäseReaktionsmechanismusDNSNobeliumMilBiskalcitratumMethylierungNucleinbasenProteineSonnenschutzmittelRegulatorgenWursthülleHistoneMethylgruppeEukaryontische ZelleGenregulationEisenGenexpressionChemische ForschungVorlesung/KonferenzComputeranimation
25:25
NobeliumReaktionsmechanismusDNSEukaryontische ZelleWasserCobaltoxideAdenosylmethioninMolekülAbschreckenStrahlenschadenFunktionelle GruppeEnzymLäsionChemische ForschungAlphaspektroskopieEisenAbschreckenWasserThermoformenStrahlenschadenFunktionelle GruppeChemische ReaktionDNSKonzentratMolvolumenVorlesung/Konferenz
26:20
DNSWasserCobaltoxideNobeliumEmail <Beschichtung>MolekülWasserChemische ReaktionDNSTrennverfahrenAdenosylmethioninFormaldehydMolekülBiochemikerinEukaryontische ZelleReaktive SauerstoffspeziesMetalloenzymChemische ForschungQuerprofilCubanReaktivitätBukett <Wein>StrahlenschadenVorlesung/Konferenz
27:06
WasserEukaryontische ZelleCobaltoxideAdenosylmethioninMolekülDNSStrahlenschadenFunktionelle GruppeAbschreckenEnzymLäsionNobeliumAtomclusterKrebsforschungReplikationMutageneseSonnenschutzmittelEukaryontische ZelleReaktionsmechanismusMolekülChemische VerbindungenAktivität <Konzentration>Vorlesung/Konferenz
27:35
SchaumweinNobeliumDNSKrebsforschungCalcineurinEnzymSonnenschutzmittelReplikationAtomclusterFunktionelle GruppeMutageneseBukett <Wein>MutagenChemische VerbindungenStrahlenschadenRadioaktiver StoffReglersubstanzFunktionelle GruppeEukaryontische ZelleKettenlänge <Makromolekül>DNSWasserEnzymAktives ZentrumSpaltflächeRäuchernSchwelteerDNS-SchädigungCobaltoxideZigaretteIdiotypSubstrat <Boden>Hope <Diamant>KrebsforschungVorlesung/Konferenz
29:27
DNSNobeliumKrebsforschungEnzymChiralität <Chemie>SchaumweinMumijoDaunorubicinReglersubstanzRäuchernFunktionelle GruppeStrahlenschadenDNS-SchädigungDNSNucleinbasenReplikationProteineSonnenschutzmittelExonucleasenMutageneseEukaryontische ZelleAtomclusterEnzymDNS-abhängige-DNS-PolymerasenSystemische Therapie <Pharmakologie>X-Pro-DipeptidaseWeinfehlerIdiotypStoffgesetzSenseChemischer ProzessElektronische ZigaretteGangart <Erzlagerstätte>GezeitenstromVorlesung/Konferenz
32:11
NobeliumComputeranimation
Transkript: Englisch(automatisch erzeugt)
00:16
I will talk to you about DNA and as you know this is the carrier of genetic information
00:22
in ourselves, contains the information that you have inherited from your parents. So it would seem immediately obvious that that must mean that DNA is very stable molecule, you have to carry information without generating mutations or other problems from
00:44
generation to generation. So if we go on to the first slide please, this is the way you usually see DNA in a university park or chemistry department, it's a double helical structure which contains the information
01:11
in duplicate by complementary base pairing between the two strands. And it does look like a very solid molecule, especially when it's made by stainless steel.
01:27
The thing is we don't have DNA molecules like this in ourselves made by stainless steel, what we have is a rather complex macromolecule and some parts of that macromolecule occurred
01:45
to me many years ago, might be more labile than we had anticipated. So if you have the next slide please. This shows one of the two strands of the DNA molecule and to investigate if this molecule
02:07
was as almost as stable as stainless steel or more labile, we just decided to do an experiment that's what biochemistry and other related topics are about.
02:21
You come up with an idea and you try to fault your own logic or see if you can confirm if your ideas are right. And to do an experiment here on stability of DNA, I and my coworkers prepared DNA radioactive
02:41
enabled in individual components like in one of the bases in guanine or in cytosine and so on. And you can do this most simply by using bacterial mutants that have defective nucleotide metabolism.
03:03
So they are dependent on the growth of you adding these bases to the growth medium. And what you can do then is add bases labeled with carbon-14 so that you radioactive the label your DNA at specific sites and then you isolate that DNA and you incubate
03:24
it for long time periods in aqueous buffers at neutral pH and follow the fate of the DNA. The question is, is it a label that you can see changes in the bases occurring with
03:43
time? If you do this at 37 degrees it becomes a very long experiment. So fortunately the DNA double helical structure is stable up to 85, 90 degrees. So you have a temporary range you can work within and there is no changes in DNA
04:03
structure or double helical structure until you come up to this cold melting temperature where the two strands separate from each other. So in that temperature range also it's known there are also called thermophiles bacteria
04:21
that can grow at 70 degrees and they have exactly the same kind of DNA and protein chemistry as we have in our cells though if you grow such a flask of cells it's so hot you can't hold it in your hand. And from making such radioactive label DNA and following its decomposition to various
04:53
temperatures we can then come up with an accurate value of how fast DNA decomposes.
05:03
The first question is does anything happen at all? And the answer is yes. The most thing you see most easily is that some of the bases in DNA, especially the purine bases, go on in on top and add them in at bottom.
05:21
They are released from DNA by cleaving of the base sugar bond. I have some problem with this one.
05:56
So this bond, the base sugar bond is cleaved at an appreciable rate and that leads
06:03
to loss of genetic information. This guanine residue is lost and the same thing happens with that. So you can say you need to correct this and the easiest would be to just have an enzyme that puts back the missing base.
06:20
But you can't do that because once you have generated a basic site here, an new information here that tells you once the guanine is lost what used to be bound to the sugar residue here. So the only way you can correctly repair something like this is by using the information
06:49
in the opposite strand of the double helix and I'll come back to that in a couple of minutes. But the first point here is could we actually measure the DNA, some decay rates
07:10
and we could do that and they are shown in the next slide please. So for loss of adenine and guanine in a single mammalian cell in one day or 24 hours,
07:27
you lose close to 10,000 bases and then there are other things happening. At a slower rate, pyrimidines are also released and there is also a bit of deamination of cytosine and mesocytosine.
07:45
That doesn't look very impressive but I should point out that these values are for double-stranded DNA which is what we experimentally work with in the cell. In a growing cell, about 2% of the DNA is actually temporarily opened up due to replication
08:06
and due to transcription, you have to process the information in DNA here. So these values are too low by a factor of 5 or 10. So deamination of cytosine is relevant and there are other things happening,
08:26
oxidation of DNA, damage to the basis of by oxidation and also there are small molecules in reactive small molecules in the cell.
08:41
This is a coenzyme, acetylnosimethionine, which is an alkylating agent, mesolating agent and that will also alkylate DNA and sites randomly where it shouldn't be alkylated. So all this background noise of DNA damage occurs all the time.
09:05
And with these high numbers, you can't lose tens of thousands of purine bases carrying genetic information every day.
09:20
So there's a dilemma here and the answer, the obvious answer to this problem is that DNA is being repaired all the time. So DNA is not perfectly stable. In a living cell, it actually turns open but at a very slow rate. But purine bases are lost and being replaced by enzymatic repair mechanisms.
09:46
And I'll say something about that now. Next slide, please. The most common lesion is loss of purine base. So here is the opposite strand of DNA with the cytosine in it and there should be a guanine here
10:04
but that has been released by hydrolysis. And this is deleterious because once you want to replicate the DNA and separate the two strands, there's information missing here. And so you better repair that while you still have a double-stranded structure.
10:24
And the way this is done is that the cell has a specific cutting enzyme, a so-called AP endonoclease that doesn't cut intact DNA but it cleaves DNA at sites where there is a missing base so you get a strand interruption here and then the rest
10:47
of this around this strand interruption are trimmed by phosphodiesterase or by an AP lyase to remove this sort of remnant of the base.
11:03
And then you have a gap of one nucleotide that's filled in by DNA polymerase which in mammalian cells is the small DNA polymerase beta that has this separate function. And finally, you can join the DNA with the joining enzyme, the DNA ligase.
11:23
So you are back to the intact DNA before you had the purination event. So this is called base excision repair and it happens all the time. At high accuracy obviously.
11:41
So next slide please. Biochemists like to take systems apart and reconstitute them and this we have done here. We identify the enzymes that were involved in this process and purify them and then we can reconstitute the process again.
12:04
There is one extra step here because I mentioned that cytosine can be deaminated to the base uracil which usually doesn't belong in DNA. It's of course in RNA but not in DNA. And there is a separate enzyme that takes uracil out of DNA and it's not a nucleus.
12:25
It's a new kind of enzyme which we call the DNA glycosylase because it cleaves the glycosyl bond, not the phosphodiester bond. So this is now the pathway to remove uracil from DNA. You remove the base and then you use the system to repair a basic site.
12:45
And with the purified enzymes, we can then reconstitute this system step by step. What we do is make oligonucleotide DNA, 41 mer, with uracil in the middle
13:01
and label it at the 5' end with P32. And then we can visualize it here. We can match it up with a complementary strand because some of these enzymes are specific for double-stranded DNA. And then when you analyze it on the gel, you run it in alkaline solution
13:22
so you can look at the individual strands. And this is what happens here. And what you see is that when you remove the uracil from DNA, you don't really see that here because removing uracil from DNA leaves a basic site. It doesn't cause a chain break by itself.
13:41
So you don't see anything here. But the DNA has now become susceptible to the special nucleus that leaves its sites where the base is missing, and then you have DNA polymerase adding one nucleotide and finally with DNA ligase, you are back to the intact double-stranded oligonucleotide.
14:03
So this is reconstitution of the system which more or less confirms that it works as we thought it would do. So I'll have the next slide, please. This is a response to unwanted hydrolytic damage to DNA.
14:24
So you might wonder is DNA damage always a bad thing? You may also know that we have mechanisms which I won't talk about today to repair radiation damage caused in DNA.
14:41
This is not what I'm talking about now. This is endogenous DNA damage. But in addition to these processes, nature has made use of the DNA repair process in higher eukaryotes because there is a big problem here.
15:01
Once we have an antibody response to nasty antigens in the environment, we need a large repertoire of antibodies. And how can I have that? You would have to use up a large proportion of your genome to code for all possible antibody chains that could interact with antigens.
15:29
What you do instead is have local genetic variability in the genes that code for antibodies in the light chains here.
15:41
There are special mechanisms that deal with these genes. So they are susceptible to genetic variation and the two main players, the deaminase AID and which deaminase usually cytosine to uracil actively.
16:01
And then we have the uracil DNA glycosylase that generates an abasic site. And with the number of processing enzymes, one can then hurt antibody genes, locally change the genetic information.
16:21
So from one gene, I can get many thousands of variations of the gene. And then some of those can be selectively triggered so that they can be used.
16:41
So this is one aspect of somatic hypermutation, which is otherwise something that can be rather worrisome because our DNA changes all the time. Sometimes I get asked, isn't this a good thing for evolution that DNA changes?
17:02
And it is, of course, up to the point, but we get bombarded with so much damage to the DNA that even a small fraction of that is more than enough to account for genetic variability in DNA.
17:23
So next slide, please. The changes I've described so far are due to hydrolysis of DNA, but there are other things that happen to DNA. Oxidative damage, as you know, we use active oxygen in much of our metabolism
17:43
and that can also interact with DNA. And the forms of damage you get then are entirely different from the forms of damage you get with waters. You have to start all over and start thinking about new repair mechanisms, how to deal with oxidative damage, though the general principle tends to be the same.
18:03
Here's one nasty change that can happen. You oxidize guanine residue at the 8th position here, so you have 8 ox of G. And that means in the DNA structure that that base flips around 180 degrees like this.
18:21
So it's upside down, and that means it is on replication paired with another purine. So you get the mutation, as you called, transversion mutation, with two purines opposite each other. And this is a very mutagenic event and you don't want that to happen all the time. And what you do is that as soon as this is formed in DNA,
18:44
there is a special enzyme that takes it out and it's another of these DNA glycosidases that leaves the base sugar bond here so that the base is released and then you have a basic site that is repaired the way I just described it.
19:01
Next slide, please. In addition to water and oxygen, there are several other things that can happen to DNA. And I think probably the most important is reaction with small reactive
19:21
molecules in the cell. And a good example of that is alkylation. In the cell, there is a small molecule, reactive small molecule called S-adenosylmethionine. And that's the coin in all trans-methylation reaction,
19:44
almost all trans-methylation reactions in the cell, because this methyl group here is very easily donated. So it's even spontaneously released and methyl acts as an alkylating agent, will alkylate at different sites in a quite non-specific way.
20:04
So it's not specific to DNA, it will alkylate DNA or RNA or protein to some extent. But if you damage a protein, there are fairly good mechanisms to degrade the protein that have been damaged and make a new protein molecule.
20:22
It's more difficult to make a new chromosome. So you have to repair this kind of event happening to DNA. And we have studied this process in some extent because there are several different sites that can be methylated in DNA.
20:43
Next slide, please. And here are a couple of these sites that you have to deal with. This is formed spontaneously in DNA in cells. 3-methyladenine is a methyl group here. Well, this is in the minor groove of the DNA double helix.
21:04
So methyl groups are not allowed because this is where polymerases travel in the minor groove of the DNA. So this is a lethal blocking lesion, has to be removed. And it's removed by yet another of these DNA glycosylases
21:20
that cleave out this damaged base. But there are other strategies that are interesting to a chemist because they hadn't really thought about this before. Here is a very mutagenic base called O6-methylguanine. And especially in higher cells, it's important to remove this
21:41
as quick as possible before you cause mutations during replication. And there is a special protein, MGMT, which recognizes this methyl group that shouldn't be there and removes it by transferring it to itself.
22:04
So you can discuss, is this an enzyme or it acts actually as an enzyme interacting with the so-called suicide inhibitor. But this means that the molecule can't turn over. You can say, why don't you just regenerate this now
22:22
and cleave this methylcysteine bond? Well, that happens to be a very stable bond. You can boil methylcysteine in hydrochloric acid and still nothing happens to it. So it would be very energy requiring
22:40
and probably require free radical chemistry to be able to regenerate this free cysteine here. And the cell gives up at this point and just makes a whole new repair protein molecule. So this is an expensive form of repair. Every time you repair this lesion, it costs the whole protein molecule. But it's rapid and it's efficient.
23:03
So that's an illustration of that energy in cells actually comes pretty cheap. I think of it like leaving the light in the bathroom. If you go out, you may or may not get around to switch it off. And there are other unusual forms of DNA repair here.
23:23
These are other sites of alkylation, which again are blocking lesions again. And we have known about this for a long time. One methyl adenine and three methyl cytosine can't be replicated.
23:42
So what do you do? Well, they are repaired somehow. And we know that in E. coli, which was the model system for much of this work, there is a special protein called ALK-B that accounts for this, which presumably is a repair enzyme.
24:01
And human cells have a counterpart to that, ALK-B homolog. A couple of homologs actually, HbH2 is the most important one. So we could clone these genes, express the protein, run it on a gel and see a beautiful protein band.
24:21
And we have lots of this protein, but we just couldn't make it do anything. And we suspected it was a repair enzyme. There was something missing in this picture. And it turned out what was missing, that this enzyme had unusual cofactors. It requires ferrous ion and also alpha ketoglutarate, a small molecule.
24:45
And biochemists usually don't add these compounds to their reaction mixes. And if they are not there, nothing will happen here. But in the presence of the correct cofactors, this protein acts as a very efficient demystilates.
25:01
And it turns out that this is a principle that nature has used in similar cases. For example, in methylation and demystilation of histones turns out to be important for regulation of gene expression
25:22
in mammalian cells. And nobody understood how you could demystilate a histone. But you use exactly the same chemistry using ferrous ion and alpha ketoglutarate. And then you can, with the correct enzyme, demonstrate to be true that you have demystilates.
25:49
Next slide, please. So I have gone through here some of the major forms of damage to DNA.
26:05
Water is a group-specific reagent and it damages DNA. That's not intuitive because water is a very weak group-specific reagent. On the other hand, it's present at a concentration of 55 molar all the time.
26:23
So even the slow reaction between water and DNA will be significant with time. There's reactive oxygen in our cells. And small cofactors that are reactive, like S-adenosylmethionine. And there are probably several others of those. We know, for example, in some metabolic pathways,
26:43
we generate formaldehyde as an intermediate or reaction product. And formaldehyde can react with DNA and cause DNA damage. And there are probably several other small reactive molecules here,
27:01
which have not yet been investigated. Just have to go back to your biochemistry textbooks and see what kind of small molecules are there in cells. And as anybody investigated, if these small molecules can react with DNA, and if so, what are the products and other special repair mechanisms
27:20
to deal with this all the time? We don't know the answer to that yet. So, last slide, please. And this is where this work is going to some extent. Question often comes up. We know that sunlight can give skin cancer, but what other compounds in the environment can give us cancer?
27:45
Well, in contrast to what you read in the daily press, we don't really live in a big sea of mutagens. So it's not a major worry. Even cosmic radiation is fairly low and accounts for the small proportion of the DNA damage we see in cells.
28:05
But what about endogenous damage? Well, it's rather difficult to evaluate that at present. We know that if you mute by a mutation, take away the enzyme that can cleave at an abasic site in DNA.
28:24
That's a lethal event to itself. So a mammalian cell that can't cleave at abasic sites is dead. With that indicates it's an important enzyme, but it doesn't tell you exactly how important it is. And the problem here is that we want to investigate that point.
28:43
There is no control group. If you want to do epidemiology, and that means you need a control group. For example, if you are worried about the possibility
29:02
that cigarette smoking and the tars in cigarette might be a cause of lung cancer, you compare chain smokers with non-smokers and you will detect the difference. And that will lead you to the right answer. But we don't have any individuals that don't get exposed to endogenous damage.
29:23
There are no cells that don't have water or active oxygen in them, so there's no control group. And when I talk with people working on smoking, they tell me that without the control group, they would have no idea that smoking is mutagenic or carcinogenic.
29:40
They just can't do the experiment because there's no control. And I think this is where we are with endogenous DNA damage. It occurs, it's fairly abundant, but exactly how important it is depends on how effective the repair processes are, and we don't have an exact answer to it.
30:01
Often the question comes, I thought about mutagenesis versus error-prone DNA replication. Well, to fix a mutation, you have to replicate the DNA. But as such, DNA replication is really very accurate.
30:21
The best experiments on this have been done by Bruce Albert's group, and they find in the T4 replication system that they set up that you just don't detect any mutations. It's less than one in 10 to the 12.
30:41
So during evolution, DNA replication has become very accurate. But of course, things can go wrong. For example, you can have a sunlight-induced alteration to a base DNA that can cause a mutation, or you might have a faulty replication factor.
31:06
Let's say you have a DNA polymerase that has become damaged, and that will now have a low fidelity when it copies DNA. Well, that probably occurs, but not in most mutations,
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because we find occasionally there are clusters of mutations, and a good explanation for that is that the replication factor temporarily has been damaged. Cells are very good proteases that can degrade damaged proteins.
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So this is the kind of thing that we are continuing this work with, and there are a number of interesting nuclear enzymes that we know very little about, like the exonuclease TEX1,
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which I think is the key factor to understand autoimmunity in mammalian cells. But that will have to wait for some other lecture, and thank you very much.