The Maintenance of Genomic Integrity

Question 1

List 3 sources of DNA damage.

Which of these is the most common?

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

List the 6 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.

*All are explain later in the lecture.

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 06 alkylguanine.
• 06 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. Dangerous!

Question 5

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

Answer 5

1 - Thymine dimers.

2 - (6-4) photoproducts.

Question 6

Describe the mechanism by which thymine dimers are 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. Dangerous!

Question 7

List 2 mutagenic substrates (seen already in previous cards) for which the repair mechanism involves direct reversal of the damage rather than removal and replacement of the damaged substrate.

Answer 7

1 - 06 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 removed 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 - see NER.

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.

*The whole process is explained in a later card

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

How is DNA polymerase eta functionally different from normal DNA polymerase?

Answer 12

DNA polymerase eta is able to replicate DNA past photoproducts.

Question 13

Describe the process of nucleotide excision repair (NER).

Answer 13

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 14

What is xeroderma pigmentosum (XP)?

What is the cause of xeroderma pigmentosum?

Answer 14

• Xeroderma pigmentosum is a condition which causes hypersensitivity to UV (sun) light.
• Patients develop many skin tumours.
• 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 15

What is xeroderma pigmentosum V?

How is it different from other xeroderma pigmentosum variants?

Answer 15

• 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 16

What is basal cell naevus syndrome?

What is the cause of basal cell naevus syndrome?

Answer 16

• 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 17

What is unscheduled DNA synthesis?

Answer 17

The DNA synthesis which occurs during the repair of DNA damage by the excision repair pathway (BER/NER).

Question 18

What is the function of BRCA1 and BRCA2?

Answer 18

BRCA1 and BRCA2 are tumour suppressor genes.

*BRCA = BReast CAncer gene.

Question 19

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

Answer 19

BRCA1 and BRCA2 repair double stranded breaks.

Question 20

List the 2 DNA repair processes that are used to repair double strand breaks.

Answer 20

1 - Non-homologous end joining.

2 - Homologous recombination repair.

Question 21

What determines whether a double strand break will be repaired by non-homologous end joining or homologous recombination repair?

Answer 21

• If BRCA1 removes 53BP1 protein from the ends of the broken DNA, non-homologous end joining will occur.
• Otherwise, BRCA2 inserts a RAD51 protein to the site of DNA damage to carry out homologous recombination repair.
• BRCA1 is also required for homologous recombination repair through interaction with 53BP1.
• Details of the repair processes isn’t needed.

Question 22

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 22

• Homologous end joining is involved in the formation of antibodies.
• It is required for VDJ recombination.
• It is ideal that homologous end joining is involved in this process because homologous end joining results in the random insertion of bases in the DNA. This contributes to the diversity of antibodies.
• NB this also means that in other instances of DNA repair, homologous end joining is particularly prone to error.

Question 23

What is PARP?

What are PARP inhibitors?

Which cells are particularly sensitive to PARP inhibitors? Why?

Answer 23

• PARP is an enzyme involved in DNA repair, and plays a role in BER and NER.
• PARP inhibitors are a type of targeted cancer drugs.
• Cells defective in homologous repair (such as those with BRCA mutations) are particularly sensitive to PARP inhibitors.
• This is because if a single strand break occurs to a template strand in DNA replication, and PARP is not present to repair the template strand via BER or NER, the single strand break becomes a double strand break when the single strand break meets the front of the replication fork.
• A cell that is unable to fix this double strand break will therefore die.

Question 24

What is the function of DNA mismatch repair?

Answer 24

DNA mismatch repair repairs copy errors made during DNA replication.

Question 25

What is a microsatellite / simple tandem repeat?

What corrects microsatellites?

What is microsatellite instability?

In which condition is microsatellite instability implicated?

Answer 25

• A microsatellite / simple tandem repeat is a tract of repetitive DNA in which certain DNA motifs are repeated. It is caused by DNA polymerase ‘slippage’.
• Microsatellites increase risk of mutation in the coding region in which they are found.
• Microsatellites are usually corrected by DNA mismatch repair.
• If DNA mismatch repair is impaired, the microsatellites will persist, resulting in microsatellite instability. A condition in which this occurs is hereditary non-polyposis colorectal cancer (HNPCC), AKA Lynch syndrome.

Question 26

What is the mutator phenotype hypothesis?

Answer 26

• The mutator phenotype hypothesis postulates that the mismatch repair defects lead to mutations in other genes, including those known to play a role in the adenoma-carcinoma sequence.
• Therefore, the increased mutation rate is the cause of further accelerated tumorigenesis.
• The mutator phenotype plays a role in tumour progression rather than initiation.

Question 27

List 4 DNA repair disorders.

Answer 27

1 - HNPCC / Lynch syndrome.

2 - Xeroderma pigmentosum.

3 - Fanconi anaemia.

4 - Bloom syndrome.

Question 28

How does the aetiology of Fanconi anaemia compare to the aetiology of BRCA1- and BRCA2-associated hereditary breast and ovarian cancer syndrome (HBOC)?

Answer 28

• In BRCA1- and BRCA2-associated hereditary breast and ovarian cancer syndrome (HBOC), only one allele of BRCA1 or 2 is mutated.
• In Fanconi anaemia, both alleles of BRCA1 or 2 are mutated.

Question 29

Which enzymes is defective in Bloom syndrome?

Answer 29

DNA helicase is defective in Bloom syndrome.

Question 30

What type of DNA damage is most commonly caused by ionising radiation?

How is this clinically relevant?

Answer 30

• Ionising radiation most commonly causes double strand breaks.
• This means that conditions that impair double strand break repair mechanisms (homologous recombination and non-homologous end joining) such as HBOC and Fanconi anaemia (which are disorders of BRCA) increase the risk of cancer formation due to ionising radiation.