DNA repair - Dr Ruddy

Question 1

What is replicative repair?

Answer 1

Replicative repair is the repairing mechanism to fix an issue due to a mistake in DNA replication

Question 2

Why do DNA miss-matches need to be repaired quickly?

Answer 2

DNA miss-matching is usually repaired but if the miss-match goes into the second round of replication the miss-match is repaired naturally (as in it no longer is a miss-match but still the incorrect base pairing) and therefore the wrong base is not detected.

Question 3

Name the three types of replicative mutations

Answer 3

1. point mutations - miss-matches, the wrong bases incorporated into the structure
2. Transition mutations - purine with purine/ pyrimidine with pyrimidine
3. Transversion mutations - these mutations tend to be more mutagenic, T to G/ A to C

Question 4

How does the cell identify which is the correct strand and which strand needs to be repaired?

Answer 4

Newly synthesised strands are not methylated so the DNA polymerase is aware of what strand is the new strand and therefore can detect the wrong strand and fix it.

Question 5

How is DNA methylated?

Answer 5

Dam methylase adds a methyl group to A in GATC sequence, but this does not occur straight away.

Question 6

Describe the process of mis-match repair in E .coli

Answer 6

Mut S protein scans for a mismatch and binds to DNA at the site. Mut S has ATPase activity and this is how it recruits MutL. Mut S recruits MutL. MutL activates MutH which nicks the DNA near the mismatch, the MutL/MutH complex is needed to nick the DNA and this complex is formed by the hydrolysis of ATP. MutH binds to the nearest GAA sequence to the mismatched pair. Helicase (UvrD) unwinds the DNA, and digests misplaced strand beyond the mismatch, this removes the mismatched base and create a gap in the process. The gap is filled by DNA polymerase III, and then sealed by DNA ligase

Question 7

What are the 5 types of DNA damage?

Answer 7

1. Spontaneous hydrolysis 
A - Deamination
B - Depurination 
2. Oxidation
3. Alkylation
4. Radiation-induced damage 
A - UV
B - Gamma
C - X-Ray
5. Chemical agents

Question 8

Why is DNA always subjected to spontaneous hydrolysis?

Answer 8

Because DNA is in aqueous solution

Question 9

Describe how Deamination mutates DNA

Answer 9

Deamination is the removal of the NH group from a base, it can happen naturally in DNA as it is in an aqueous solution. This causes mutations as it changes the DNA base pairing rules and in the case of the GC bond it makes it a lot weaker and this has many negative effects on DNA.
Deamination of cytosine to Uracil causes base pairing with Adenine
Deamination of Adenine to hypoxanthine causes base pairing with cytosine
Deamination of guanine to xanthine causes base pairing more weakly to cytosine as there are now only 2 hydrogen bonds.

Question 10

Describe how Depurination mutates DNA

Answer 10

Spontaneous hydrolysis of the N-glycosidic bond to produce a deoxyribose without a base. This type of hydrolysis releases the base and a puridic ribose (ribose sugar without a base) is attached.

Question 11

Describe how Oxidation mutates DNA

Answer 11

Oxidation of DNA is caused by Reactive oxygen Species (ROS) such as O2-, H2O2, or OH. Guanine is especially susceptible to ROS and when guanine is oxidised it forms oxoG which can base pair with both C and A.

Question 12

What three mutations can the base excision mechanism repair?

Answer 12

Deamination
Depurination
Oxidation

Question 13

Describe how Base Excision repairs DNA

Answer 13

A specific glycosylase enzyme recongises that the certain base it is specific to should not be incorporated into that part of the DNA, the enzyme cleaves at the glycosidic bond and removes the incorrect base. An endonuclease then cleaves the 3’OH and then an exonuclease removes the other side 5’ bond so what the whole sugar and phosphate is removed. This is done so that it creates a gap that DNA polymerase can then put in the correct nucleotide.

Question 14

What is the fail-safe glycosylase?

Answer 14

If the base excision repair mechanism does not work before the next cycle of replication occurs, then there is a fail-safe glycosylase.
Its best to explain the example in terms of an oxoG error being incorporated.
Replication occurs before the oxoG is repaired and then oxoG pairs with adenine. A specific glycosylase recognises the incorrect incorporation of A and removes it, now the cell has a second chance to repair the original oxoG mutation.

Question 15

Describe how Alkylation would mutate DNA

Answer 15

This is where methyl of ethyl groups are transferred to bases. Guanine is a good example as the double bonded oxygen (O6) in guanine is susceptible to alkylation. The mutated version of guanine often pairs with T. O6-methylguanine often pairs with thymine resulting in GC to AT mutation.

Question 16

Describe how Direct repair is used to repair alkylation

Answer 16

Methyltransferase removes CH3 and transfers it onto a cysteine residue, this can only occur once for each methyltransferase. The enzyme methyltransferase recognises the methylguuanine, binds to it and takes on the methyl group itself on one of the cysteine residues in its structure and thus repairing the DNA. This is an expensive system as the enzyme cannot get rid of the methyl group once it is bound, so the enzyme can only be used once.

Question 17

Describe how DNA can be mutated by Radiation

Answer 17

UV - strongly absorbed by nucleoside bases, resulting predominantly in thymidine dimers. DNA polymerase cannot pass and DNA replication stops.

Question 18

What is a thymidine dimer?

Answer 18

The formation of a cyclobutane ring between two adjacent thymines is a thymidine dimer.

Question 19

Describe how direct repair can be used to repair DNA damaged by radiation

Answer 19

The damage is removed in situ via a process of photo reactivation. DNA photolyase enzyme recognises the dimer and binds to it but in order to repair it it needs light energy to remove the cyclobutane ring and restore the two thymine bases independently. DNA photolyase uses energy form light to break covalent bonds in the cyclobutane ring.

Question 20

Describe 3 types of chemical mutagens and how they mutate DNA

Answer 20

Base analogues - Sufficiently similar to bases to be incorporated into the DNA but they do not base pair correctly and so cause mutations. They may not be similar enough in structure to allow replication to occur either, 5-bromouracil is similar to thymine and incorrectly base pairs with guanine
Tautomers - molecules with the same molecular formula but the bonds are differently placed, these structures are not the same as resonance structures. These also miss-pair and create mutations
Intercalating agents - These are flat polycyclic molecule which bind to the equally flat purines and pyrimidines. Thought to cause DNA polymerase to add a base opposite the intercalated agent or to skip a nucleotide. E.g. ethidium bromide or acridine orange. The DNA polymerase gets confused when it encounters these agents and either skips or repeats bases in the sequence creating duplication or deletion mutations.

Question 21

What method usually repairs thymidine dimers or chemcial mutagens?

Answer 21

Nucleotide Excision, this is where a complex of proteins removes nucleotides to either side of the lesion and repairs the gaps. The proteins do not recognise a specific lesion, but they recognise a kink in the DNA double helix. This method is an extension of base excision as it removes more then just the base it removes the whole nucleotide or a segment of nucleotides

Question 22

Describe how nucleotide excision repairs mutated DNA

Answer 22

UvrA in complex with UvrB scans and detects DNA distortion, they bind to the DNA at the site. After binding the UvrA dissociates.
UvrB melts the DNA around the distortion to separate the strands and forms a bubble
UvrC is recruited by UvrB and this nicks the DNA 5’ and 3’ to the lesion
UvrD is a DNA helicase and it unwinds the DNA and in this instance removes the segment of DNA that contains the misstake.
DNA polymerase I synthesises the new strand and DNA ligase seals the gap

Question 23

Describe how nucleotide excision is different in humans

Answer 23

The proteins are homologous and have slightly different names;
UvrA - XPC
UvrB - APA/APD
UvrC - XPG
XPG is the protein that nicks the 3’ region in humans, another protein called ERCC1 or XPF nicks the 5’ region.
XP stands for Xeroderma pigmentosum and this is a disease. Patients cannot be exposed to UV light as they cannot repair the thymidine dimers that this causes as they cannot perform nucleotide excision, A mutation in any of the proteins above causes this disease.

Question 24

Describe transcription coupled repair

Answer 24

If a mutation has not been repaired and the gene starts to be transcribed then this repair mechanism comes into play. The RNA polymerase will not be able to pass the mutated region of the DNA. The RNA polymerase acts as a sensor for DNA damage because the RNA polymerase not being able to pass the region triggers the nucleotide excision repair mechanism to start and the DNA is repaired.

Question 25

Describe how Ionising radiation causes DNA damage

Answer 25

Ionising radiation includes X-Rays and Gamma rays, both induce double stranded breaks by directly damaging the DNA backbone, which cannot be replicated. Ionising radiation also induces the formation of ROS.

Question 26

How does the cell usually repair double stranded breaks?

Answer 26

Using double stranded break (DSB) pathways which utilise the sequence information from sister chromatids, the correct information is read from the sister chromatid and used to repair the double stranded break.

Question 27

If no replication has occurred yet and there are no sister chromatids for a sequence to be read off how does the cell repair double stranded breaks?

Answer 27

Has two methods the first is NHEJ which stands for homologous end joining - This method basically takes two pieces of DNA and shoves them back together. The downside to this mechanism is that sequence information is lost at the broken ends and this makes this method highly mutagenic. This is the principle pathway of repairing DS breaks in higher organisms
The second is translesion repair - When DNA has replicated and the mutation has persisted, DNA polymerase cannot replicate past the mutation. The DNA polymerase just leaves a gap in the mRNA. This gap in the mRNA is repaired by translesion DNA polymerase. However, this polymerase needs a template to fill the gap in, as there is no template the enzyme just guesses which bases to put in, it is random selection. One in 4 times it will get it right, again this is highly mutagenic.

Question 28

describe NHEJ in DSB repair

Answer 28

Ku70/80 (a heterodimer protein) recognises the broken ends and binds, it recruits DNA-PKcs and this protein then recruits Artemis. Artemis then in turn recruits Ligase IV which ligates the two DS breaks together.

Question 29

What are the two prominent methylation systems in E. coli?

Answer 29

Restriction modification (defence mechanism to distinguish foreign DNA)
GATC methylation by Dam methylase as part of repair system during DNA replication.

Question 30

What are the two types of methylation systems in eukaryotes?

Answer 30

• Addition of methyl groups at CpG sites on the DNA

- Cytosine methylation found in mammalian and plant DNA, but not in worms, flies or yeast

Question 31

Where is methylation most common in eukaryotes?

Answer 31

At the CpG sequences which where made by the deamination of cytosine and methylcytosine.

Question 32

Describe how CpG islands are made

Answer 32

After replication the C and G bases on a daughter strand are both correct. If a cytosine is methylated (usually means the gene is turned off) and then it is deaminated (through a mutation) then a thymine miss pairing occurs and this is not repaired with the same efficiency as an unmethylated cytosine deamination into Uracil. This therefore reduces the amounts of G and Cs in the daughter strands, evolution, therefore, will naturally lead to more A and Ts in DNA compared to G and Cs. CpG islands are thought to persist as they are active genes and therefore not methylated and therefore a deamination mutation will be repaired back to a C.

Question 33

What are the two ways to methylate DNA?

Answer 33

Maintenance methylation - This is when the patterns are just copied from what was there already
De novo methylation - Brand new methylation occurs and this is important during embryogenesis and development.

Question 34

Describe maintenance methylation

Answer 34

The newly synthesised daughter strand has not been methylated as soon as it has been made, it is hemi-methylated DNA. Hemi-methylated DNA is recognised by the enzymeMethyltransferase, this enzyme recognises the methylated sites on the parent strand and basically copies them over to the daughter strand.

Question 35

Describe De novo methylation

Answer 35

After fertilisation all the methylated patterns are removed and the methylation process starts again with De Novo methylation. De novo methyltransferases are directed to the DNA by DNA binding proteins and once at the DNA the enzymes will recognise specific sequences in the DNA that should be methylated, then it will methylate them.

Question 36

What enzymes are responsible for DNA methylation?

Answer 36

Maintenance methylation - Dnmt1 (DNA methyltransferase 1) in mice
De Novo methylation - Dnmt3A and Dnmt3B
Mutations in Dnmt1 in mice means the mice will die by day 11. Mutations in Dnmt3A/B results in neural tube defects.

Question 37

What are the three mechanisms for gene silencing?

Answer 37

1. Un-methylated DNA adopts an open conformation which is accessible for proteins such as transcription factors. Histone proteins bind to DNA and the DNA wraps up around them making chromatin. This wrapping condenses the DNA and makes it less accessible. Methylated DNA condenses more and inactivates a gene.
2. Methyl groups can physically impede the binding of transcription factors
3. Proteins called Methyl-CpG-binding proteins preferentially bind methylated DNA through their Methyl CpG binding domains (MBD) and inhibit transcription .

Question 38

How does the Dnmt1 protein recognise hemi methylated DNA?

Answer 38

Methyl-CpG-binding proteins bind to the methylated DNA and then interact with the Dnmt1. There are three families of these types of proteins classified according to how they recognise methylated DNA;

• Methyl CpG binding proteins
• SRA-SET and RING finger proteins
• Zinc finger proteins

Question 39

Describe passive demethylation

Answer 39

This is nonenzymic, the methyl groups just passively fall off.

Question 40

Describe active demethylation

Answer 40

Directly removing the methyl group perhaps by excising it through a repair mechanism

Question 41

Describe what imprinting is

Answer 41

Methylation patterns are erased after fertilisation with the exception of imprinted genes. These gene escape the mechanism of demethylation. Imprinting is the inheritance of an allele in a parent-of-origin specific manner. Imprinted genes are often clustered together on the same chromosome.
Insulin-like growth factor 2 (IgIf2) gene is an imprinted gene, paternal allele is expressed and maternal allele is suppressed. The promoter region of the IgIf2 gene is methylated in oocytes but not in sperm. For most genes it doesnt matter which allele from which parent is turned on or off but in imprinted genes it does.

Question 42

Describe why imprinted genes exist in clusters

Answer 42

They exist in clusters as they share regulatory elements, an ICR and an enhancer. The regulatory system can only work one way, so if one of the genes is active the other is suppressed and vice versa.
In males the IgIf2 gene is on and the H19 is off. In males the ICR is heavily methylated and this methylation can extend to the 5’ region of H19. This heavy methylation inhibits the binding of CTCF and allows the enhancer to influence the transcription of IgIf2 and switch it on, as H19 is blocked the enhancer cannot turn on H19.
In females the ICR is not methylated so CTCF (an insulator protein) can bind and this blocks the action of the enhancer and therefore it cannot access the IgIf2 promoter which is then turned off but it can access the H19 promoter and therefore it is switched on.

Question 43

What diseases can a mutation in the ICR lead to?

Answer 43

Beckwith-Weidemann syndrome (BWS)

Silver-Russel syndrome