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BIOL2010 > DNA repair > Flashcards

DNA repair Flashcards

1
Q

DNA damage can be done by what 3 things?

A
  • Replication errors
  • Spontaneous damage (occurs naturally in cells e.g. oxidative phosphorylation in mitochondria)
  • environmental damage (e.g. smoking, UV damage)
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2
Q

Damage can be what and what do these words mean?

A

Damage can be Endogenous or Exogenous

Endogenous: within an organism

Exogenous: outside of an organism

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3
Q

Is all DNA damage a mutation?

A

No mutation is the worst thing that can happen from DNA damage

DNA damage is distinct from mutation

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4
Q

What is a mutation?

A

A change to the nucleotide sequence in DNA

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5
Q

DNA repair

A
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6
Q

misincorporation of nucleotides during replication leads to mutation. Can you explain this using a diagram?

A
  • Changes in DNA structures can lead to incorrect nucleotide base pairing also just incorrect pairing can lead to this
  • Mutations cannot be repaired
  • Write the mutation location as shown above in arrows
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7
Q

With nucleotides e.g. purines and pyrimidines what are the mutations that can arise and what are these?

A

Can either be Transition or Transverse mutations

  • Lead to missense and nonsense mutations (not frame shift mutations)
  • Transition: purine converts to purine or pyrimidine to pyrimidine
  • Transversion: purine changed to pyrimidine or the other way around
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8
Q

Whats a Missense mutation?

A

When a single base change occurs in DNA sequence, so when protein ends up with different AA

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9
Q

Whats a Nonsense mutation?

A

Mutation introduces a stop codon which means protein sequence may not be fully made. Again, this is a single base change

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10
Q

From the causes of DNA damage, which ones cause a covalent change and a non-covalent change?

A

Non-covalent: Replication errors

Covalent: Spontaneous/ environmental damage

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11
Q

What are some replication error mutations?

A
  • Mismatches/ Tautomerisation
  • Strandslippage/ intercalation
  • Transposition/ Aberrant recombination
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12
Q

What are some spontaneous/ environmental damage mutations?

A
  • Hydrolysis (depurination, deamination)
  • Oxidation/ Alkylation/ Bulky adduct formation
  • Radiation (UV, X-rays, gamma rays)
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13
Q

Are replication errors a natural part of DNA replication?

What is the error rate in DNA pol and after proof reading?

A

Yes

The error rate in typical DNA pol is 1 in 105 nucleotides (107 after proofreading)

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14
Q

The usual base pairings for nucleotide bases are AT and GC, what are the mismatched pairs?

Whats affected in them?

A

The “wobble pairs” are AC and GT as these are both mismatched.

The AC has 2 H bonds as AT would

the GT has 2 H bonds but the GC would normally have 3 H bonds making it unstable

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15
Q

Draw adenine

A
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16
Q

Draw guanine

A
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17
Q

Draw cytosine

A
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18
Q

Draw thymine

A
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19
Q

Draw uracil

A
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20
Q

What is Tautomerisation?

A

alternative H positions

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21
Q

Whats the usual form of T

Tell me about the other form?

A
  • In 1 in 1,000 residues the T is often found in enol form
  • Enol form of T can form a base pair of G. This enol TG is stable as forms 3 H bonds
  • Know that imino A (another form of adenine) pairs with C
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22
Q

What is insertions due to strand slippage?

Where do they occur?

A

They occur at repeat sequences

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23
Q

What are deletions due to strand slippage?

Where do they occur?

A

They occur at repeat sequences

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24
Q

Insertions due to intercalation are exhibited by what?

Give an example

A

Flat/ aromatic compounds e.g. ethidium bromide

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25
Q

Disruption to the replication machinery can lead to what?

A

Insertion mutations e.g. causing framshifts

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26
Q

Here we have a nucleotide stacking between base pairs. What can this lead to?

A

One of nucleotides stacking between base pairs. This slightly unwinds DNA and increases its length slightly (as shown in red circle). Can lead to incorrect nucleotides being added leading to frameshift

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27
Q

What are insertions due to transposition?

A
  • Or alternatively, due to aberrant recombination events between sequences or chromosomes
  • Transposons can jump out of a region of DNA and then go into another site of genome due to transposons
  • Retrotransposons goes through an intermediate before being incorporated into DNA
  • Sometimes transposons can jump into a DNA sequence an interupt it
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28
Q

66% of genetic mutations that develop into cancer are caused by what?

A

Simple replication errors

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29
Q

Chemical modifications to DNA are caused by what?

A

By endogenous (from metabolism) and exogenous agents (including UV and ionising radiation)

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30
Q

What do chemical modifications involve in DNA ?

A

Structural alterations to the double helix, which can be minor or more pronounced

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31
Q

DNA damage can lead to mismatches and are therefore mutagenic, in the same way as replication errors. What can this induce?

What does this lead to?

A

This can induce single or double strand breaks (DS breaks are the most mutagenic)

This can lead to blockage/ stalling of the replication and transcription machinery e.g. polymerases, transcription factors etc

They can influence gene expression by altering chromatin architecture

32
Q

What does the loss of base by hydrolysis (depurination) involve?

What is it more likely with?

Is it mutagenic, if so why?

A
  • Involves spontaneous hydrolysis of the glycosidic bond between a purine and its sugar, leading to the formation of an abasic site (apurinic; AP site)
  • 20 times more likely with purines; 4 times more frequently in ssDNA
  • Loss of sequence information so mutagenic; and increased susceptibility to strand breaks
  • Water breaks glycosidic bond, leading to loss of DNA base
  • Depurination occurs more reading with purines
  • Apurinic site is where base is lost (due to hydrolysis by water)
  • 20% more likely with purines
  • More likely with ssDNA
33
Q

What is deamination?

What is the by product?

When it occurs with A and G what is generated?

A
  • Water hydrolyses the bond that attaches the amine
  • The by product is ammonia but it isn’t toxic at these concentrations
  • Also occurs with adenine and guanine, generating hypoxanthine and xanthine, respectively
34
Q

Why does DNA contain T, but RNA contain U?

A

So that the body is able to detect cytosine deamination products

35
Q

Note about deamination

The deamination of 5-methylcytosine produces T, which is much harder to detect, so methylated CpG steps are underrepresented in the eukaryotic genome

Promotors regulated by CpG steps (epigenetic change)

CpG= CG the p is the phosphate group between the C and the G base

A
36
Q

Where are Reactive oxygen species (ROS) generated?

A

In the mitochondria (electron transport chain) as well as by ionising radiation

37
Q

What is base oxidation often accompanied by?

A

Strand breaks

38
Q

Base oxidation by ROS

A
39
Q

Base oxidation by reactive nitrogen species (RNS) converts what?

A

Converts adenine to hypoxanthing (H)/ inosine which pairs with C instead of T

e.g. Nitric oxide NO- (endogenous factor, environmental pollutant)

40
Q

In the presence of O2, what can NO be converted to?

A

NO2- and N2O3, which can lead to the deamination of amine-containing bases

41
Q

Whats base alkylation?

A
  • Transfer of methyl, ethyl or other alkyl groups from nitrosamines and other DNA alkylators, usually to nitrogen or oxygen of base, e.g., s-adenosylmethionine (endogenous methyl donor)
  • Alkylating agents often used in chemotherapy (bifunctional so cross-link strands)
42
Q

Give an example of a chemical that does bulky adduct formation?

A

Aflatoxin B

43
Q

What was Aflatoxin B first metabolised by?

A

Cytochrome P450 in the liver, which generates reactive epoxide intercalator

44
Q

Explain base dimerisation caused by UV exposure

A
  • UV exposure can cause cross linking in DNA
  • occurs frequently in pyrimidines. In presence of UV light can become cross linked (CPDs)
  • T-T- dimer really distort DNA so easy to detect and repair at a later stage
45
Q

Explain the Ames test and how it was used to examine the mutagenic potential of a compound?

A
  • Exploiting bacteria salmonella that has a modified version of a gene in it
  • The gene normally encodes for the protein that make histidine (histidine synthase)
  • Gene has frameshift do can’t produce functional protein
  • Has to provide histidine in growth medium in order for it to survive
  • If doesn’t contain histidine, then it won’t grow
  • If incubate with mutagen, which mutates DNA in cells, it can affect frameshift mutation in the histidine synthase gene, this turn gene back on, assay as you would in absence of mutagen, as protein has erected the bacteria will survive on a medium that doesn’t contain histidine
46
Q

What are the estimated rates of DNA damage per human cell per day?

A
  • Depurination 10,000
  • Deamination 600
  • Oxidative base damage 2000
  • Alkylated bases 5000
  • Intra-strand cross links 10
  • Single strand breaks 50,000
  • DNA double-strand break 10 (caused by ionising radiation)
  • Total DNA damaging events per cell per day: 60,000
  • Total DNA damaging events per cell per hour: 2,500
  • Estimate 1013 - 1014 cells in human body
  • ~ 3 x 1012 DNA damaging events during this lecture
47
Q

What are the main DNA replication errors you need to know?

A
  • Mismatches: GT and CA
  • Tautomer’s
  • DNA damage; Abasic sites, C –> U, 8-oxo-G, 6-O-MeG, CPDs
48
Q

What are the mechanisms for repairing replication errors and DNA damage?

Give examples of some errors they can repair?

A
  • Direct reversal (DR) – acts directly on modification (alkylation / UV)
  • Base excision repair (BER) – removes damaged base (deamination / oxidation / alkylation): C–> U, 8-oxo-G and 6-OMeG can be fixed by this method
  • Nucleotide excision repair (NER) – removes damaged nucleotide(s) (UV / other)- this method can fix CPDs
  • Mismatch repair (MMR) – removes mismatched base (replication errors)
  • Double-strand break repair (DSB) - joining of broken DNA ends (X-rays)- very mutagenic and hazardous
  • Translesion synthesis – bypass damage
49
Q

Why is it often possible to repair damaged DNA?

A

Because the information is stored on both strands of the double helix

50
Q

Tell me about direct reversal (in human system) and what enzyme is involved and what it does?

A
  • Irreversible stoichiometric reaction (“suicide enzyme”)- only does the process once
  • MGMT is the enzyme involved- name of enzyme tells you what it does
  • MGMT flips DNA bases out of helix and into its active site. Once found the lesions, it takes off the methyl group and sticks it into its active site
  • Methylated form of MGMT is a transcription activator, upregulates synthesis of other repair proteins
  • Confers resistance to alkylating drugs used in chemotherapy
51
Q

Explain DNA photolyase (taking place in E.Coli system)

A
  • Requires light to do this process
  • Flavoprotein containing two light-harvesting co-factors
  • In the presence of visible light (blue; 370 nm) FADH radical acts as an electron donor to break the pyrimidine dimer; found in some sunscreens
  • Not a suicide enzyme like the last one
52
Q

What is Base excision repair (BER)

A
  • DNA glycosylate breaks glycosidic bond between base and sugar which generates an AP site
  • AP endonuclease notices missing base, cleaves backbone/ removes sugar
  • DNA Pol I/ ligase gap fills and seal nick
53
Q

What is base excision repair (UDG) found in humans and E. Coli?

A
  • UDG removes uracil and base
  • Flips out DNA bases in active site (looks for uracil along DNA and flips out bases once found)
  • Uracil can enter active site and thymine cannot
54
Q

What is base excision repair with MutM/Y/T - found in E. Coli ?

A
55
Q

Whats nucleotide(s) excision repair (NER)?

A
56
Q

Whats nucleotide excision repair with UvA/B/C/D in E. Coli ?

A
57
Q

How is nucleotide excision repair in eukaryotes similar to that in E.Coli, but what is different?

A

Similar but more complex process (involves 25+ proteins)

  • XPC (UvrA) recognises lesion
  • XPA and XPD (UvrB) opens and binds the DNA
  • RPA (single-stranded binding protein) stabilises the open complex
  • ERCC1-XPF and XPG (UvrC) create incisions and remove oligonucleotide
58
Q

Is the Single stranded segment in NER (eukaryotes) longer ot shorter to that in E. Coli?

A

The single-stranded segment is longer (24-32 nucleotides)

59
Q

In eukaryotes, what does the NER complex also take part in?

A

Transcriptional coupled repair

60
Q

What is NER is eukaryotes recruited to?

A

An RNA polymerase stalled at the site of a lesion, focusing on the repair of proteins on genes being actively transcribed; also involved the transcription factor TFIIH

61
Q

With Mismatch rapir (MMR) what is the error rate that polymerases exhibit?

How does proof reading change this error rate?

DNA replication proceeds with a error rate of what in nucleotides?

Therefore what is MMRs error rate?

A
  • Polymerases exhibit an error rate of 1 in 105
  • 3’-5’ proofreading activity increases this to 1 in 107
  • DNA replication proceeds with error rate of 1 in 1010 nucleotides
  • Therefore, MMR provides an error-reducing process of 1 in 103
62
Q

What does MMR involve?

A

Repairing the main GT, GG, AC and CC mismatches

63
Q

What is MMR similar to but how is it different?

A

Has a similar excision pathway to BER and NER but results in the removal of much longer stretchs of nucleotides

NB: the system must correct the mismatch accurately; that is, it must replace the misincorporated nucleotide in the newly synthesised strand and not the correct nucleotide in the parental strand

64
Q

Tell me about base mismatch repair or MutS/L/H in E. Coli

A
65
Q

With double stranded break repair, what are the two main pathways?

A
  • Homologous recombination
  • Non-homologous end joining (NHEJ)
66
Q

Whats homoglous recombination?

A

It can be used to exchange sequences between similar or identical DNA moleucles (e.g. a sister chromosomes in a diploid cell)

67
Q

What happens early in the cell cycle before two sister chromosomes have been generated?

A

NHEJ

68
Q

Whats NHEJ?

A

Non-homologous end joining protects and processes the broken ends and then joins them together, but usually results in small deletions and is therefore mutagenic

69
Q

What are the 7 proteins that NHEJ involves and what do they do?

A
  • Ku70 and Ku80 bind the broken ends
  • Recruit the kinase DNA-PKcs and the endo/exonuclease Artemis to cleans up/process ends
  • Finally, DNA ligase IV joins the ends whilst in complex with XRCC4 and Cernunnos-XLF
  • Ubiquitous in eukaryotes but rare in prokaryotes
70
Q

What are Translesion polymerases?

A

Non-processive, template-dependent polymerases that synthesise across a blockage, usually by incorporating nucleotides in a manner independent of base pairing (thus very error prone)

However, some are specific for certain lesions (e.g., Pol η mediates error-free bypass of lesions induced by UV irradiation)

71
Q

Tell me about Translesion polymerases (regulation by SOS response) in E. Coli?

A
72
Q

Whats the p53 gene?

A
  • Transcription factor that regulates the expression of genes involved in growth arrest, DNA repair, and apoptosis (tumour suppressor protein)
  • Levels of cellular p53 are increased during DNA damage and cellular stress
73
Q

Name two diseases linked to DNA repair?

A
  • Xeroderma pigmentosum (XP)
  • Hereditary non-polyposis colon cancer (HNPCC)
74
Q

Whats Xeroderma pigmentosum (XP)?

A
  • Individuals show dry, parchment-like skin (xeroderma) and many freckles (pigmentosum)
  • ‘Children of the night’- as cant come out in daylight
  • Increased sensitivity to UV light
  • 1000-fold increased risk of skin cancer
  • Due to inherited defects in one of eight distinct genes responsible for components of the NER complex
75
Q

Whats Hereditary non-polyposis colon cancer (HNPCC)?

A
  • Individuals exhibit a predisposition to colon cancer (2-3% of all colon cancer cases)
  • Due to defects in the human equivalents of the MutSHL mismatch repair system (MSH2 and MLH1)
  • Leads to the accumulation of mutations throughout the genome and over time genes controlling gene proliferation become affected
  • Only therapy is removal of the colon
76
Q

Other diseases linked to DNA repair

A
77
Q

Summary of lecture 7 and 8

A
  • DNA is susceptible to replication errors (mismatches, tautomerisation) as well as spontaneous and chemical damage (hydrolysis, oxidation, alkylation, radiation, etc.)
  • Can lead to mutations, disrupt DNA/RNA synthesis, or lead to apoptosis and cell death
  • Various proteins have evolved to repair DNA damage that act alone or in combination (e.g., direct reversal, BER, NER, MMR, NHEJ and recombination)
  • In some cases damage can be skipped by error-prone translesion polymerases
  • Cells are capable of upregulating the expression of various proteins to initiate repair (SOS response) or halt the cell cycle (e.g., p53)
  • Similar mechanisms exist in both prokaryotes and eukaryotes
  • Mutations to genes that enocde DNA repair proteins lead to disease and highlight their importance

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