Ls3 - Lecture 9

Front 1

What is a mutation?

Back 1

Any permanent change in the DNA.

Front 2

What causes mutations?

Back 2

Replication errors: Because polymerase is not 100% foolproof, it has its 5’ to 3’ exonuclease activity so that if it recognizes it’s made a mistake, it can take a step back to correct it. If it doesn’t catch that, then it’s a replication error. The poly has introduced the wrong nucleotide and hasn’t figured that out. There will be another repair pathway that comes in and takes care of this.

Spontaneous mutations: Always going on in body due to natural circumstances

Radiation and mutagens: External mutagens such as radiation and chemicals that damage DNA. A chemical that is going to increase a mutation rate is considered a mutagen.

Front 3

What is a non-permanent mutation called?

Back 3

A lesion

Front 4

Are all mutations bad?

Back 4

No. In order to evolve, we have to have mutations. If they are favorable, they get selected for. This is how we get diversity.

Advantageous Mutation: Mutation that benefits the organism

Deleterious Mutation: Mutation that harms an organism.

Mutations can also be introduced into DNA to study how proteins function etc.

Front 5

Mutagens come from intrinsic and extrinsic factors

Back 5

Intrinsic: From within the cell (naturally occuring)

Extrinsic: From outside the cell

Front 6

Guanine and Intrinsic mutagens example

Back 6

Guanine and different points of guanine are susceptible to many mutations. Our general cell metabolism can produce reactive oxygen species. We can do oxidative damage to our own cell as a by-product of metabolism. This is going on in the mitochondria (powerhouse of cell). The mitochondria generate a lot of these free radicals by just digesting and breaking down the molecules of what we are consuming. Because of this, a restrictive caloric diet can lead to longevity because you consume less, so you generate less of these free radicals that cause aging and bad effects.

Front 7

Guanine and Intrinsic mutagens example #2

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Also applicable to Guanine. Natural hydrolysis in the cell could also damage the DNA bases as well as chemicals that add on bulky adducts to them(Called alkylation by chemicals).

Front 8

Guanine and Extrinsic mutagens example

Back 8

Also applicable to Guanine. UV light damage

Front 9

Spontaneous base damage by hydrolysis

Back 9

When we have spontaneous mutations based on hydrolysis, they can take the form of deamination or depurination.

Front 10

Deamination of cytosine to uracil

Back 10

Deamination by hydrolysis of cytosine to uracil. This can happen at anytime in the DNA. This is responsible for seeing uracil bases in DNA. In order for this Uracil to go back to becoming a Cytosine, they often are methylated in the cell (not considered mutation).

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Depurination of guanine

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Depurination of guanine. Just lose to nitrogenous base of guanine. This is considered an abasic site. The most common base loss is the depurination of guanine.

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Deamination of 5-methyl cytosine to thymine

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Exactly as name suggests.

Front 13

Base damage by oxidizing agents

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These are all the natural and unnatural ways that cells can generate free radicals.Free radicals are what create the oxidizing agents that will do base damage.Just understand that the bases can be oxidized and be damaged. Comes from radiation, air pollution, UV damage and the mitochondria.

Front 14

UV Damage - Thymine Dimers

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UV damage is what causes two thymine nucleotides to dimerize (two things that are working together) if they are close together. Thymine dimers create a large bulge in the DNA backbone and this prevents DNA replication from occurring. So if a DNA is trying to replicate a thymine dimer, it can’t and it will not know what to do. To avoid cellular damage, we must fix this. Different proteins and enzymes will recognize the bulge (kink) in the DNA. DNA has a very specific helical structure and specific angles so when we have mismatches, dimers or missing abase, we have different mechanisms to counteract those.

Front 15

Intercalating agents

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Agents that wedge themselves into the DNA and can cross-link the DNA. These are considered bulky adducts that are added onto the DNA. This prevents normal replication of the cells. The most important intercalating agent is Ethidium Bromide. Intercalating agents cause insertions or deletions.

Front 16

DNA damage done by chemotherapeutic agents

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We use DNA damaging agents specifically with chemotherapy. Chemotherapy is a way to treat cancer. A lot people who receive chemo will develop other cancers because cells become highly damaged. We have many reactions like hair loss, cell damage etc. because chemo targets cells that rapidly divide because they are the ones that will be bound to the DNA and prevent replication and this will introduce mutations into the cells.Cancer cells rapidly divide, but so does hair and stomach cells so this is why we have these side effects.

Front 17

Base Analogs

Back 17

Things that resemble nucleotides in the cell. They can sometimes make their way into our DNA, but they must be removed.

Front 18

Most common example of base analogs?

Back 18

One of the most common examples of base analogs is the kemo-tautomer of 5-bromo-uracil. This is a modified base meaning that it has a different function other than functioning in the DNA as a nitrogenous base. When this goes into it’s enol form, it can base pair with guanine. So if the 5-Bromouracil is floating around while DNA is replicating (DNA poly is moving along, thenucleotides are being added in) if the enol form is around, the DNA poly can use this and mistake it for a Cytosine. So what would be a GC base pair is now the modified 5-Bromuracil attached to the guanine. This is a point mutation.

Steps:We have a normal AT base pair, we have a 5-Bromouracil that can enter its different form and base pair with Guanine. If we were to replicate after this,it would become a permanent lesion. However we still have some time to correct this.

Front 19

Failure to correct lesions leads to mutations

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Mutations in cells that form gametes will be passed on to progeny (diseases or phenotypic differences).

Mutations in cells that don’t form gametes (form in the somatic cells),can interfere with gene expression or replication and lead to formation of tumors and cancers, or speed up aging. These are external mutagens and won’t be passed on to offspring.

Front 20

What is one effect if we fail to correct the lesion and it becomes a mutagen?

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We have an altered base that shouldn’t be there (x). We separate the two blue strands (parental strands) and replicate the DNA. For the top one, a C was added to the opposite strand because the (x) best matched with the C. The bottom side is normal. They will keep separating and the process will continue to match the opposite strands. So what should have been an AT pair is now a G.If this occurs in an important region of a gene, a single mutation can ruin the function. A lot of times, this occurs in ‘junk DNA’ that doesn’t really have a function. In this DNA, we have a lot of repetitive sequences and only 1% of our genome is turned into proteins.

Front 21

DNA replication isn't perfect

Back 21

When we have the correct base pairings, they have a specific angle. So when a mutation occurs, we have enzymes that can recognize these mismatched base pairs because they alter the angle of the DNA and cause a kink.

Front 22

Explain this pic

Back 22

Here we have a poly that wanted to replicate the DNA. So the parent strands separate (grey). If, for example, the A accidentally gets paired with a G, the angle is disrupted. On the other strand, the correct nucleotide is added. If this isn’t fixed, when we separate the strands, they will become the new parental strands and DNA poly can fix the mistake from before by adding the correctly paired nucleotide and effectively get rid of the bulgeΠT

Front 23

What are the types of mutations?

Back 23

Single base/small changes: Point mutations, Insertions and Deletions

Large Scale (chromosomal) changes: Translocations, Duplications/Insertions and Deletions

Front 24

Single base/small changes

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Point Mutations: A single change in a nucleotide (A become a G). Only affects 1 nucleotide

Insertions:Nucleotide could accidentally be added

Deletions:Nucleotide could accidentally be deleted.

Front 25

Large Scale (Chromosomal) changes

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Translocations: BCR Gene example, look in book

Duplications/Insertions: Large portions of the chromosome could get duplicated (a region in a chromosome could get doubled).

Deletions:Large portions of the chromosomes could get deleted.

Front 26

Point mutations are classified as either transitions or transversions

Back 26

When you go from a purine to a purine, or a pyrimidine to a pyrimidine, it’s called transition. The number of aromatic rings stays the same.

When you go from a purine to a pyrimidine, it’s called transversion. The number of aromatic rings stay the same. Transversions are more likely to result in further mutations downstream.

Front 27

What is the effect on small mutations on protein sequence?

Back 27

The genetic code is the structure of the nucleotide sequence. Grouped by 3’s. 3 nucleotides will make a codon. A codon codes for a specific amino acid during translation. So we have the genetic code which has a reading frame, and the frame that it’s in is the set of 3’s. We can’t just start at a point and count by 3’s, there is a specific grouping. Within the sequence of nucleic acid that becomes a protein, we have a start codon that encodes for methyanine. This is always the first amino acid that is incorporated into the protein. We will also have a stop codon, which will stop it and stop translation. Making slight changes in the genetic code will cause a different amino acid to be made,however, some of the codons won’t change.

Front 28

What will happen if you change the reading frame?

Back 28

Changing the reading frame can cause a premature stop, which will stop the codon early.This is also a frame shift mutation, because anything that shifts the genetic code is called a frameshift mutation. Frameshift mutations are almost always deleterious and nearly always disrupt the function of the protein. So as long as you add/delete under 3 nucleotides, it will cause a frameshift mutation. Sometimes you can have an insertion or deletion that is 3 nucleotides at a time, which is really lucky and this would be less likely to affect the function.

Front 29

Point Mutations

Back 29

Silent Mutation, Missense Mutation and Nonsense Mutation

Front 30

Silent Mutation

Back 30

Mutation that doesn’t affect the protein at all. For example, we could have UAU could accidentally by changed into UAC, but wouldn’t make a difference because they both encode for Tyr.

Front 31

Missense Mutation

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Whena nucleotide is change and causes a different amino acid to be encoded.

Front 32

Nonsense Mutation

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Mutation that introduces a STOP codon by changing a single nucleotide.If this happens at any point within the reading frame, we will have a truncated form of a protein. Will interrupt the proteins function

Front 33

Insertions and Deletions

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Frameshift Mutation: Changes reading frame and changes downstream.

Front 34

Back 34

D. Frameshift Mutations.

Frameshift mutation because Point Mutations are single changes in one of the nucleic acids. It’s not adding/removing them, just a change in the nucleic acids. Point mutations are often caused by DNA poly or deamination. Don’t disrupt the reading frame and don’t cause frameshift

Front 35

There are many DNA repair pathways to protect the genome, what are they?

Back 35

Mismatch Repair: Fixes replication errors (errors which happen during replication such as a wrong nucleotide added) Photoreactivation: This will fix thymine dimers. We are the only organisms that has the enzyme to fix this.

Base Excision Repair: Takes out a single base that is damaged from hydrolysis,analog being incorporate etc.

Nucleotide Excision Repair: Used to fix thymine dimers and any sort of bulky addition to DNA.

Double-Stranded Breaks: Fixes breaks in the double strand by using homologous chromosome as a template or by stitching the chromosome back together.

Front 36

DNA Mismatch Repair

Back 36

First have an enzyme that will recognize that there is a mismatch. Scans DNA to make sure if it has a mismatch by analyzing the angles or bulges. After it recognizes it, it will bind to the site. It then recruits other factors. An endonuclease will come in and make a nick in the phosphodiester backbone in the region of the lesion. Then an exonuclease will ‘chew’ away some of the DNA surrounding the lesion. Next we have DNA Poly that will come in and synthesize the region. Finally, ligase will seal the nick because DNA Poly can’t. (5 essential enzymatic functions).

Front 37

How does MMR know which was the original strand/base?

Back 37

We need to know which was the parental strand, the correct base and the wrongly incorporated base. As soon as the DNA is synthesized, enzymes will go in and methylate it. By methylating DNA in specific places, unmethylated DNA will be able to be distinguished and they can start chewing this one up. There is a brief period of time where the DNA is not methylated, and if we recognize the lesion and its on the unmethylated strand, that has to be where the problem was because the methylated strand was the parental strand. So DNA is methylated AFTER replication and the old strand has methylation, the new one doesn’t. So endonuclease will recognize which side isn’t methylated and cut the strand at the point of the lesion. If it’s not methylated there is no way to determine which side it is on.

Front 38

Nucleotide Excision Repair

Back 38

For bulky mutations (thymine dimers) as well as intercalating agents that are large

For humans, this is the only way to repair the thymine dimers because we don’t have the photoreactivation

Recognizes and removes bulky lesions

Similar repair pathways found in all organisms, from bacteria to humans.

Here we have enzymes that recognize the bulky lesion, then bind it. They will create a nick around the bulky distortion. They can tell which side the DNA is on is because it will intercalate on one side or the other. It can tell which base has something bulky attached to them. There is no need for methylation here. Helicase will unwind the DNA and will use ATP energy to remove DNA by the unwinding ability. DNA Poly will come and seal gap, then Ligase will come and seal nick. In this case, helicase performs the endo/exonuclease activity.

Front 39

Difference between MMR and NER?

Back 39

MMR will only repair single base changes from replication. NER will repair bulky lesions and doesn’t need methylation. NER will repair thymine dimers and large bulky adducts.

Front 40

Base Excision Repair

Back 40

Mechanism that removes single modified bases (modified by deamination, base analogs orketo-enol etc.). Many different enzymes that recognize specific common modifications have been identified such as Deaminated C to Uracil, Deaminated 5-methyl C to thymine and Depurination

Front 41

BER: DNA Glycolases

Back 41

Enzymes that recognize base modifications and remove them. A glycosylase will severe the glycosidic linkage between the sugar and the base of a nucleotide. It removes the base because it’s the base that gets modified so you can make your job easier if you leave the sugar and fix the base. Will leave an abasic site.

Glycosylases scan the minor groove, facilitate base flipping

Front 42

BER Pathway

Back 42

Let’s say we have our normal Cytosine that was deaminated into a Uracil(most common deamination). An enzyme, DNA glycosylase will recognize the Uracil and cleave the Uracil base leaving just the sugar which is an abasic site. The phosphodiester backbone is still intact. Once we have this abasic site, an endonuclease will come in and cut the backbone. This allows DNA poly to come in and add only 1 nucleotide (which in this case is the correct base Cytosine).Lastly, DNA ligase will come in and seal gap.

Front 43

MMR & RBE Differences?

Back 43

MMR is from replication errors, RBE fixes modified bases.

Front 44

Double-Stranded Break Repair

Back 44

Generated by ionizing radiation (X-rays) or oxygen, free radicals, radiation, reactive oxygen species made in the mitochondria. To repair, uses Non-homologous end joining (NHEJ) and homologous recombination (HDR).

Front 45

Non-homologous end joining (NHEJ) & pathway

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Considered a mutation inducing repair mechanism. Just tried to paste chromosomes back together. Will result in insertion or deletions of points in chromosomes so it won’t go back to the point it was because as soon as it’s severed, it’s subject to exonuclease degradation, so some info will be lost forever.

Pathway:We have a double stranded break that is repaired, and a series of enzymes that bind near the edges of these breaks and seal the DNA back together. But this almost always leads to insertions and deletions and introduce a frameshift mutation.

Front 46

Homologous Recombination (HDR)

Back 46

Error free replication mechanism. This will utilize a homologous chromosome as a template to synthesize the region around the breaks to make sure we retain all the info.

Pathway:Go through this pathway to repair effectively. We have another set of enzymes that bind the edges of the double stranded break and they will cleave away more of the DNA that was necessary so that we have an overhang that’s long enough to undergo strand invasion. So we have another chromosome that has the same genetic information. It finds a homologous region in that other chromosome through the strand invasion then we use this as a template to fill in the gap.So the reason we tear more than needed, is so that we have an overhand long enough to find where its sequence homology is on the sister chromatid. Once it does that, then it knows whatever is on the opposite side is what should be repaired in the DNA double strand break.

There could be point mutations in the sister chromatid used as the template so if they are slightly different, we are going to replicate the change directly into our strand that we tore.

Pathway repeated: We have a double stranded break in our chromatids. We have series of enzymes that bind where the break was and chew away extra. Re-synthesis happens and make full DNA into exactly what is should be.

Front 47

If all else fails...

Back 47

Use Translesion Synthesis. This is if you have damaged DNA, normal DNA poly won’t know how to replicate this correctly. Other poly’s won’t care and will be able to synthesis DNA with bad lesions just so the cell could survive. This introduces mutations but makes them perfect and saves the cell.

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Front 49

Some diseases associated with what we've been talking about?

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Xerodonum Pigmenosum

Huntington's Disease

Cockayne Syndrome

Fragile X Syndrome

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Xeroderma Pigmenosum

Back 50

Condition where we are deficient in one of any NER Enzymes (end/exo, dna poly etc) that can’t repair damage done by UV radiation. You can’t repair thymine dimers. People are extremely sensitive to sun exposure.

Closer Look: Thymine dimer is induced and will prevent DNA replication. As you increase UV light, XP cells won’t be able to replicate because they have so many mutations. However, normal cells can replicated but they have an increase in radioactive labeling because you added a radioactive nucleotide.

Front 51

Huntington's Disease

Back 51

Involves a protein that is nonfunctional but takes a while to manifest. We have an expanded repetitive sequence (often difficult to replicate cuz so long). In Huntington’s Disease, the protein Huntington has many CAG repeats. If you end up moving past the threshold of having a certain amount of repeats, we have the manifestation of the disease. Going past the threshold makes the protein nonfunctional. It can retain up to about 35 of the repetitive sequences before it becomes nonfunctional. You can be born with over 30 of these repeats and manifest the disease young. Depending on how many your born with will determine when you manifest the disease. Detected by PCR.

Trinucleotideexpansion: caused by unequal crossover during meisos

Front 52

Fragile X Syndrome

Back 52

In the region that encodes a gene, we have other parts of the dna that recruit RNA poly that will transcribe DNA into RNA. We also have other transcription factors that will control the genes expression. We have a region that precedes the gene that has a repetitive sequence. Normally we should have a certain amount of repeats. If we have more copies of the repeat, we get degradation of the chromosome. Only occurs in males on their 1 X chromosome. Most common source of mental retardation. When we have regions that expand in front of the gene region, sometimes they are methylated. Methylation of cytosines helps control genes expressions. Normally methylation is associated with a decrease in gene expression. CG is more likely to be methylated.