L10 - The Maintenance of Genomic Integrity

Question 1

How can DNA be damaged?

Answer 1

1 - Copying errors during DNA replication (most common)

2 - Spontaneous depurination

3 - Exposure to carcinogenic agents (e.g. UV light, tobacco and ionising radiation)

Question 2

What are the major types of DNA repair?

Answer 2

Generally, repair involves either:

1 - Direct enzymatic reversal of the DNA damage

or removal and replacement:

2 - Base excision repair (BER)

3 - Nucleotide excision repair (NER)

4 - Homologous recombination

5 - Non-homologous end joining

6 - DNA mismatch repair

Question 3

What is 7-methyl-guanine?

What might produce 7-methyl-guanine?

Answer 3

• 7-methyl-guanine is a methylated guanine nucleotide that is a biomarker for some cancers when found in the urine
• It results in distorted DNA when DNA is replicated, causing cell death
• It might be produced spontaneously or as a result of alkylating drugs

Question 4

What is ethyl methane sulphonate?

Answer 4

• Ethyl methane sulphonate drug (used in DNA repair studies) that induces a mutation by alkylating guanine, producing O6 alkylguanine.
• O6 alkyl guanine pairs with thymine rather than cytosine
• The modified guanine is then replaced with an adenine, resulting in an overall change (mutation) from G-C to A-T
• This does not result in cell death, but is still mutagenic

Question 5

What is the major form of damage caused by UV light?

Answer 5

1 - Thymine dimers

2 - (6-4) photoproducts

Question 6

How are thymine dimers formed by UV light?

Answer 6

• Adjacent thymines are covalently linked by UV light
• This causes a distortion in the DNA, resulting in difficulties at DNA replication
• This does not result in cell death, but is still mutagenic

Question 7

Which mutagenic substrates have a repair mechanism that involves direct reversal of the damage rather than removal and replacement of the damaged substrate?

Answer 7

1 - O6 alkylguanine is repaired by alkyl transferase, which removes the alkyl group from the guanine

2 - UV induced thymine dimers are repaired by monomerisation (breaking the covalent bonds), which occurs by the combined action of visible light and photolyase

Question 8

List 3 characteristics of base excision repair (BER).

Answer 8

1 - Operates on either double stranded or single stranded DNA

2 - Recognises specific mutagens using a range of glycosylase enzymes

3 - Removes and replaces single bases

Question 9

Describe the process of base excision repair (BER).

Answer 9

1 - The altered DNA base is excised in free form by a DNA glycosylase

2 - The resulting abasic site is corrected by an apurinic endonuclease

3 - Addition of new nucleotides is carried out by DNA polymerase and DNA ligase

*Doesn’t work for large adducts such as thymine dimers

Question 10

List 4 characteristics of nucleotide excision repair (NER).

Answer 10

1 - Only operates on double-stranded DNA because NER requires a template

2 - Non-specific, therefore recognises distortions rather than specific adducts

3 - Removes and repair large adducts, e.g. thymine dimers

4 - Very efficient and error free

Question 11

What is daughter strand gap repair?

Answer 11

• If a large adduct (e.g. thymine dimer or photoproduct) is present at DNA replication, there will be a gap in the new (daughter) strand of DNA
• Daughter strand gap repair is a tolerance mechanism, whereby the gaps are repaired after DNA replication by way of DNA polymerase eta, but the large adducts remain (and are ‘tolerated’)
• The thymine dimers are removed later from the double stranded DNA by NER

Question 12

Describe the process of nucleotide excision repair (NER).

Answer 12

1 - XPC and XPE proteins recognise DNA caused by large adducts (e.g. by a thymine dimer)

2 - XPA and TFIIH are recruited to verify the DNA damage

3 - XPB and XPD act as helicases to unwind the DNA surrounding the DNA damage

4 - XPF and XPG excise the damage and surrounding nucleotides

5 - DNA polymerase resynthesises the DNA across the excised region

Question 13

What is xeroderma pigmentosum (XP)?

What is the cause of xeroderma pigmentosum?

Answer 13

• Xeroderma pigmentosum is a condition which causes hypersensitivity to UV (sun) light
• Patients develop many skin tumours (in areas exposed to light)
• It is caused by a defect in NER (due to a defect in one of the XP proteins). They therefore cannot remove large adducts formed in the DNA (such as thymine dimers)

Question 14

What is xeroderma pigmentosum V?

How is it different from other xeroderma pigmentosum variants?

Answer 14

• XPV is a variant of XP in which DNA polymerase eta is mutated
• This means that daughter strand gap repair is impaired, but NER isn’t

Question 15

What is basal cell naevus syndrome?

What is the cause of basal cell naevus syndrome?

Answer 15

• Basal cell naevus syndrome is a condition which causes sporadic formation of basal cell carcinomas
• It is caused by a mutation in PTCH1, which is a gene that codes for the receptor for sonic hedgehog
• Sonic hedgehog is a secreted molecule implicated in the formation of embryonic structures and in tumorigenesis

Question 16

What type of DNA damage is repaired by BRCA1 and BRCA2 genes?

Answer 16

Double strand breaks

Question 17

Describe the mechanism of homologous recombination repair.

Answer 17

• When both strands broken, exposed ends of DNA protected by factors e.g. 53-BP1
• HRR requires both BRCA1 & BRCA2 -> BRCA1 involved in removal of 53-BP1 & BRCA2 essential for intracellular movement & activity of Rad51
• Release of Rad51 triggered by DNA damage by phosphorylation of Rad51 or BRCA2
• Rad51 recombinase enzyme coats ssDNA to form nucleoprotein filament that invades & pairs with homologous DNA duplex, initiating strand exchange

Question 18

How is non-homologous end joining different to homologous recombination repair?

Answer 18

• Non-homologous end joining more straight forward, but more prone to error than HRR
• Rad51 independent and BRCA2 not required

Question 19

Which of the two double strand break repair mechanisms is involved in the formation of antibodies?

How is this repair mechanism involved?

Why is it ideal that this repair mechanism is involved in antibody formation as opposed to the other?

Answer 19

• The break in double stranded DNA leads to the projection of proteins protecting the exposed ends, such as DNA PK, that are then joined together by DNA ligase enzymes
• Errors occur in this process, as any gaps in the DNA are just filled with random nucleotide sequences
• This is tolerated, as this process is the basis for V(D)J recombination in immunoglobulin and TCR production, where the addition of random nucleotides is desired as it will increase diversity of the immune response

Question 20

What is PARP?

What are PARP inhibitors?

Which cells are particularly sensitive to PARP inhibitors? Why?

Answer 20

• PARP repairs single stranded DNA breaks all the time spontaneously
• If PARP is inhibited, when the replication fork meets a single stranded break it will form a double stranded break - normal cells will be able to repair this using homologous recombination repair
• Cells defective in homologous repair are usually sensitive to PARP inhibitors, therefore potential treatments of BRCA1/BRCA2 deficient cells could involve the use of PARP inhibitors to initiate apoptosis in these tumour cells

Question 21

What is the function of DNA mismatch repair?

Answer 21

DNA mismatch repair repairs copy errors made during DNA replication

Question 22

What is microsatellite instability?

Answer 22

• Insertion-deletion loops arise as a consequence of polymerase ‘slippage’ during replication -> slippage causes gains or losses in repetitive DNA = microsatellite instability (MSI)
• Mutations in mismatch repair genes increases MSI -> can lead to further mutations in other genes containing microsatellites -> tumour progression not initiation
• MSI frameshift mutations associated with hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome)

Question 23

What is the mutator phenotype hypothesis?

Answer 23

• Microsatelite mutations can lead to a mutator phenotype, in which additional mutations are conferred to the cell, accelerating tumourgenesis
• This mutator phenotype has a role in tumour progression rather than initiation, but is known to lead to mutations in the adenoma-carcinoma sequence