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BMS242 Cell and Molecular > L6 - Damage Repair and Recombination > Flashcards

L6 - Damage Repair and Recombination Flashcards

1
Q

What four ways can damage occur to DNA

A

Deamination
Depurination
Pyrimidine Dimer Formation
DNA breaks

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2
Q

What three types of chemical reactions would cause DNA damage

A

Oxidation, hydrolysis and methylation

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3
Q

What way would oxidation damage the DNA

A

At C=C double bonds (gain of oxygen or loss of hydrogen)

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4
Q

How would a hydrolysis reaction damage the DNA

A

Splitting a bond using water - can liberate from amino (NH2 groups)

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5
Q

Describe how methylation would damage the DNA

A

Nitrogen (N) can be methylated to NH2 sponatenously on any of the four nucleotides

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6
Q

What bases are purines

A

A or G

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7
Q

What bases are pyrimidines

A

C or T

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8
Q

Describe how a depurination occurs

A

Nitrogenous base removed from the deoxyribose sugar - using water

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9
Q

Describe how deamination occurs

A

Change from cytosine to uracil (will pair with A)

Uses water and releases ammonia

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10
Q

Describe how depurination can cause mutation

A

Nucleotide is missing therefore there is a deletion in one of the strands

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11
Q

Describe how deamination can cause mutation

A

C–>U so now pairs with A, point mutation of a base pair in a strand

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12
Q

Describe how a deamination may be repaired

A

By base excision repair

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13
Q

What is the enzyme involved in base excision repair which recognises the damage

A

Uracil DNA glycosylase

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14
Q

In base excision repair what is the sugar removed with

A

Apurinic/apyrimidinic endonucleases

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15
Q

In base excision repair what is the phosphate removed using

A

Phosphodiesterase

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16
Q

In base excision repair the removal of the sugar and phosphate causes –>

A

Formation of primer:template junction –> act of DNA polymerase
Nick is the sealed using DNA ligase

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17
Q

What occurs during pyrimidine dimer formation

A

When two adjacent pyrimidines are able to form a covalent linkage

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18
Q

What does pyrimidine dimer formation lead to

A

Arrest of DNA replication
Misreading by polymerases
Unable to complementary base pair with purines

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19
Q

Describe the process of nucleotide excision repair

A

Excision nucleases cleave the single strnaded DNA with the defect
DNA helicase removes the damaged segment
Formation of primer template junction leads to activation of DNA polymerase then the nick is sealed by DNA ligase

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20
Q

What disease is as a result of nucleotide excision repair deffects

A

Xeroderma Pigmentosum

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21
Q

What are the 7 xeroderma pigmentosum genes

A

XPA XPC XPD XPF XPG

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22
Q

What do the xeroderma pigementosum genes all encode

A

Proteins which are involved with nucelotide excision repair

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23
Q

What are the E.coli homologues of the xeroderma pigmentosum genes

A

UvrA UvrB UvrC UvrD

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24
Q

What is nucelotide excision repair targetted to

Why is this the case

A

DNA sequences which are being actively transcribed

By the physical coupling of RNA polymerase to the repair mechanisms

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25
Q

Why are ds breaks dnagerous

A

Mean that large fragments of chromosomes can be lost

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26
Q

What are the two methods of repairing a double strand break

A

Non-homologous end joining

Homologous recombination

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27
Q

Why is non homologous end joining not be the ideal way to repair DNA

A

No specificity - so likely to cause mutation

Can cause deletions

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28
Q

Describe the process of non homologous end joining

A

Accidental double stranded breaks
Loss of nucleotides due to degredation
End joining gives a deletion mutation

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29
Q

Describe the process of homologous recombination

A

Double stranded break
Resection at the 5’ end so can be used as a template and anneal with the undamaged chromosome
Binding protein RecA promotes strand invasion of the undamaged template by one strand from a damaged DNA molecule that acts as a primer
Formation of heteroduplex facilitates templated synthesis of the one stand
Newly synthesised strand from its template and reaneals to partner strand - 2nd strand synthesis and two nicks sealed by ligation

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30
Q

What are BRCA2, ATM and Fanconi Anaemia all examples of

A

Genes involved in homologous recombination

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31
Q

What are BRCA2 mutations associated with

A

Breast, ovarian and prostate cancer

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32
Q

What are ATM mutations associated with

A

Ataxia telangiectasia - leukameia and lymphoma

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33
Q

What occurs once homologous recombination is inneffective

A

The cell becomes critically dependent on other mechanisms of repair

34
Q

If a cell that has a mutation in the genes involved in homologous recombination is then treated with an inhibitor for base excision repair
What is the clinical relevance of this

A

Too much DNA damage and the cell will die - synthetically lethal
Can be used to treat cancer

35
Q

What drugs are specific inhibitors of poly ADP ribose

A

Olaparib and lumpaza

36
Q

What does homologous recombination machinery mediate

A

Genetic recombination in meiotic cells

37
Q

What is the function of SPO11 in homologous recombination

A

Makes the initial clevage

38
Q

What is the function of Mre11 in homologous recombination

A

Resection at the 5’ end

39
Q

What does RecA mediate

A

Strand invasion

40
Q

What does homologous recombination result in

A

Formation of the double holliday junction

41
Q

What is the resolution of the double holliday junction if only internal strands are broken and rejoined

A

Then no recombination

42
Q

What is the resolution of the double holliday junction if both internal and external strands are cut

A

Recombinattion

43
Q

Recall the purine bases

A

Adenine, guanine

44
Q

Recall the pyrimidine bases

A

Thymine, cytosine, uracil

45
Q

What are the four main types of DNA damage

A

Deamination, depuration, pyrimidine dimers, DNA breaks

46
Q

What are the three main methods of DNA repair

A

Base excision repair, nucleotide excision repair, homologous recombination

47
Q

What chemical modifications can lead to DNA damage

A

Hydrolysis, oxidation or random uncontrolled methylation

48
Q

What is the most frequency types of DNA damage

A

Hydrolytic depurination and deamination of bases

49
Q

What happens when cytosine is deaminated and what are the downstream effects of this

A

Converted to uracil. This will still pair with guanine but during replication the guanine will be replaced with an adenine, leading to a nucleotide substitution from CG–>TA

50
Q

Explain how deamination of cytosine is achieved

A

The NH2 group attached to the purine ring of cytosine at position 4 is replaced with a carbonyl group

51
Q

Explain the process of depurination

A

The carbon to nitrogen bond between the carbon position one in the deoxyribose sugar and the nitrogen in the purine ring is hydrolysed. This releases the base and results in the loss of a base pair, nucleotide deletion, in one of the daughter DNA molecules produced during replication

52
Q

Which DNA damage(s) is base excision repair used for

A

Deamination and depurination

53
Q

Explain the process of DNA excision repair

A

The damage base is first removed by DNA glycosylase which cleaves off the inappropriate base(s). The deoxyribose sugar is then removed by an enzyme known as apurinic/apyrimidinic endonuclease. Lastly the phosphate is removed by the phosphodiesterase enzyme. This leads to a ssDNA break in the polynucleotide chain. This gap is then recognised by DNA polymerase which then extends over this gap which acts as a primer template junction. Finally, DNA ligase uses ATP hydrolysis to provide the energy required to seal the nick

54
Q

What are pyrimidine dimers and what causes them

A

Pyrimidine dimers are caused by the covalent linkage of benzene rings in two adjacent pyrimidine bases. This is usually caused by UV light

55
Q

Which base is primarily affected by UV light and more prone to form dimers

A

Thymine

56
Q

What is the results of the formation of pyrimidine dimers

A

This arrests DNA replication or can cause mis-reading of the DNA sequence by DNA polymerase

57
Q

Nucleotide excision repair is only used to repair pyrimidine dimers, T or F

A

F – it is used to repair a variety of different types of DNA damage including pyrimidine dimers

58
Q

Pyrimidine dimers can only occur between identical adjacent pyrimidine bases, T or F

A

F – it can be the same pyrimidine or different ones (i.e. T-C, T-T, C-C)

59
Q

Explain the process of nucleotide excision repair

A

An excision endonuclease enzyme cleaves the single strand of the DNA containing the defect by cleaving either side of the dimer etc. This creates a polynucleotide fragment from the DNA molecule that contains the defect which is then removed by DNA helicase. DNA polymerase then extends the primer template junction created to replace the excised sequence. This is followed by resealing of the nick mediated by DNA ligase.

60
Q

Xeroderma pigmentosum is a disease caused by defective nucleotide excision repair machinery, what is the effect of this on patients

A

XP renders patients extremely sensitive to sunlight-induced skin cancer

61
Q

How many different genes have been identified as being responsible for xeroderma pigmentosum, give some examples

A

7 different genes including XPA, XPC, XPD, XPF and XPG

62
Q

What are the products of genes encoded by the genes mutated in xeroderma pigmentosum

A

These genes encode proteins that participate in the nucleotide excision repair pathway

63
Q

What is significant about the function of the XP genes

A

Their function is tightly coupled and targeted to regions of the genome that are highly transcribed. This acts as a surveillance system to signify regions of the genome that are transcribed. RNA polymerase required for transcription is physically coupled to the DNA repair machinery

64
Q

Uvr genes are homologues of the XP genes found in yeast and are also transcription coupled, T or F

A

T

65
Q

dsDNA breaks are often caused by non-ionising radiation, T or F

A

F – they are caused by ionising radiation

66
Q

Why are dsDNA breaks especially dangerous

A

Large fragments of chromosomes can be lost

67
Q

What two methods are there of repairing dsDNA breaks

A

Non-homologous end joining, homologous recombination

68
Q

What can make DNA molecules susceptible to dsDNA breaks

A

If the DNA has already suffered a ssDNA break due to base excision repair or nucleotide excision repair. This may weaken the DNA structure

69
Q

What is meant when we refer to non-homologous end joining being quick and dirty

A

Non-homologous end joining is a far from optimal repair mechanism. It is carried out quickly but in itself, can induce mutations

70
Q

Explain the process of non-homologous end joining

A

The ends of the double stranded break are rendered flush with loss of bases via degradation from the ends of the strands. These flush ends are then ligated together. However, this leads to a loss of DNA sequence due to the degradation that occurs prior to ligation.

71
Q

Homologous recombination also isn’t a perfect repair mechanism, T or F

A

F – homologous recombination provides a perfect repair of dsDNA breaks and is an accurate and preferred method of repair

72
Q

What does homologous recombination rely on

A

Using the intact DNA sequence information in the undamaged homologous chromosome

73
Q

Explain how homologous recombination is mediated

A

Firstly, the 5’ ends are resected by exonuclease to create single strands that can be used to prime DNA synthesis when annealed to a template strand from the complimentary chromosome. The DNA-binding protein RecA promotes strand invasion of the undamaged template molecule by one strand from the damaged DNA molecule that acts as a primer. This forces complimentary binding with the same sequence in the undamaged sister chromatid. This forms a heteroduplex structure between the dsDNA helix of the sister chromatid and the single stranded sequence from the damage DNA molecule. This facilitates templated DNA synthesis of one strand by DNA polymerase. DNA polymerase synthesises across the damaged region by reading information out of the undamaged sister chromatid. The newly synthesised DNA then dissociates from its template and re-anneals to its original partner strand allowing second strand synthesis and formation of a pair of staggered singles stranded nicks. These nicks are then ligated by DNA ligase

74
Q

Give an example of a gene involved in homologous recombination that when mutated can cause cancer

A

BCRA2 – causing breast, ovarian and prostate cancer

75
Q

What happens in patients with mutations in genes needed for homologous recombination

A

Homologous recombination is defective and thus these cells become critically dependant on other pathways for correct DNA repair i.e. base excision and nucleotide excision repair

76
Q

How can this defective recombination pathway seen in some cancers be harnessed as a cancer treatment

A

Combined inactivation of homologous recombination by mutations and the inhibition of base or nucleotide excision repair by anticancer drugs make cells that suffer DNA damage unable to make adequate repairs to their DNA and die. This is known as synthetic lethality.

77
Q

Homologous recombination is required to mediate genetic recombination and diversity created in meiosis, T or F

A

T

78
Q

Explain how a Double Holliday Junction is created during genetic recombination in meiosis

A

Two chromatids exist with different alleles for two genes (AB and ab). Spo11 endonuclease makes an initial cleavage in chromatid to create a dsDNA break. Mre11 then carries out a 5’ resection to create a targeted dsDNA break with a 3’ overhang in the DNA molecule. RecA then mediates strand invasion by this strand on the other chromosome creating a heterocomplex/triple helix structure. DNA polymerase then synthesises across the gap between the end of the 3’ overhang from the invading strand and the 3’ end of the other chromosome. The 3’ end is then relegated with the original strand to create a double holliday junction whereby the invading strand from one helix is forced into interactions with the complimentary strand in the helix of the other chromatid and vice versa.

79
Q

How many ways can the Double Holliday Junction be resolved

A

2

80
Q

Explain how different resolutions of the Double Holliday Junction account for whether homologous recombination occurs or not

A

If the internal strands are broken and re-joined than the same DNA strands are broken and re-joined at each junction and recombination is not achieved as each chromatid will contain the same sequence of alleles it did prior to cleavage. If external and internal strands are broken and re-joined then different DNA strands are broken and re-joined at each junction. Hence recombination is achieved. This recombination of allelic forms results in chromatids with different combinations of alleles (Ab and aB)

BMS242 Cell and Molecular (26 decks)

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