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genetics 2 > DNA damage and repair > Flashcards

DNA damage and repair Flashcards

1
Q

provide some numbers to convey just how much DNA we have

A

32 trillion cells (32,000,000,000,000)
Each cell contains 2 metres of DNA
3 billion DNA base pairs per cell
9.6x1022 bases in total

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2
Q

an individual cell can experience how much DNA damage?

A

up to 1 million DNA damage events per day
for one cell
and we have 32 trillion

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3
Q

give some common types of DNA damage?

A

sequence changes:

reactive oxygen species
thymine dimers
deamination
depurination

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4
Q

reactive oxygen species - what does it do, how does it come about?

A

often are by-products of cellular metabolism (mitochondria)
Or generated by radiation exposure

ROS react with DNA bases, changing their chemistry (forming oxidised bases)

Disrupt base pairing
Can also attack the DNA backbone, causing breaks

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5
Q

what are thymine dimers?

A

Occur when covalent links form between neighbouring thymines

Inhibits DNA replication

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6
Q

Deamination - what is it and what problems does it cause?

A

The loss of the amine (NH2) group from cytosine bases, changing them to Uracil (U) – not found in DNA

Affects DNA replication, polymerase can mistake uracil for thymidine (T)

Causes and G-A switch in the new sequence (incorrect)
Can be repaired via base excision repair (BER)

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7
Q

what is depurination, what kind of damage does it cause?

A

it is the spontaneous loss of adenine or guanine bases,
5000 bases are lost per cell per day

Causes a loss of genetic information, decreases stability in the region – generally not good

Can be repaired via base excision repair (BER)

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8
Q

Single strand breaks - how serious are they, how do they occur?

A

Cell very good at fixing this
Can become a problem if the cell does not fix this (e.g. can become a DSB)

Super common, can occur in some repair mechanisms

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9
Q

Double stranded breaks - what causes them, and what can they result in?

A

Ionising radiation and carcinogens can cause DSBs
Can be caused by unresolved stalled replication forks
note - also occur naturally in meiosis and recombination

Multiple breaks can lead to large genome rearrangements
Halts growth, if not resolved apoptosis should be triggered
Can cause impaired tissue function if damage is significant

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10
Q

failure to repair DSBs can lead to what? include an example

A

Deletions of certain regions entirely, or translocations

E.g. Chr 9 and 22, Abl and BCR now next to each other - myeloproliferative neoplasms - leukaemia (Philadelphia chromosome)

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11
Q

what are four types of mutations?

A
  1. Silent - same Aa coded for
  2. Missense - changes one amino acid
  3. Nonsense - PTC - Premature stop codon. Can be ‘dominant negative’ - not doing it’s function but interfering in the pathway
  4. Insertions and deletions - can also cause an early stop codon, cause a frameshift and disrupt the whole sequence
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12
Q

mutations have three classifications?

A

Pathogenic - cause disease

Benign - not a problem

Unclassified - no idea what it does. Is it a problem

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13
Q

are mutations all bad?

A

no, some just cause variation which drives evolution

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14
Q

how many mutations are required to cause disease? explain the multiple hit theory

A

some diseases are caused by a single nucleotide mutation, like sickle cell disease

cancers often require multiple mutations -
a single mutation in a cell can increase the likelihood of another occurring, accumulation of 2-8 mutations can be enough for a cell to become cancerous. E.g a mutation in a DNA repair gene would lead to more mutations

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15
Q

give examples of exogenous sources of DNA damage

A

Non-ionising radiation (UV)

Ionising radiation (X-ray, gamma rays etc)

Thermal damage (burns)

Alkylating agents (tobacco smoke, chemicals)

Chemotherapy drugs (Cisplatin)

Viruses (Influenza virus A2/HK/68)

Plant/fungal toxins (Aflatoxins)

Excess hormones (Oestrogen HRT)

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16
Q

give some examples of endogenous sources of DNA damage

A

Replication errors (Fork collapse, metaphase issues, synthesis mistakes)

Complex DNA structures (Hairpins/repetitive sequences/ RNA hybrids)

Reactive oxygen species (metabolism products)

Depurination (loss of A and G bases)

Deamination (C to U conversion)

Telomere shortening (End replication problem)

Deficient DNA damage response (incorrect DNA repair)

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17
Q

UV -
Causes?
general exposure and effects?
repair?

A

primarily thymine dimers

UV is non-ionising, normal UV (UVA and UVB) exposure can cause 100,000 DNA damage events per day

It’s the primary cause of skin cancer

Usually quickly repaired by base or nucleotide excision repair

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18
Q

what is ionising radiation, the different kinds etc…

A

Ionising radiation is energy released from the disintegration of atoms (electrons get knocked off)

Travels as -
waves (gamma or X-rays)
Or as particles (Alpha, beta or neutrons)

Differ greatly in their energy, range of travel and ability to penetrate materials (goes a, b, x-ray, gamma, neutron)

causes -
Double strand breaks (DSBs)
Single strand breaks (SSBs)
Energy from radiation breaks covalent bonds
A loss of DNA bases

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19
Q

X-rays -
are?
cause?
used in?

A

Made of pure energy (photons)
Lower energy than gamma rays
Can cause DNA breaks but cells repair most of this

obviously X-rays, and CT scans

20
Q

Gamma rays -
what are they?
how dangerous and damaging are they?

A

pure energy - photons again

they are dangerous in that they easily penetrate the skin and clothing, they can completely pass through the body

they are extremely damaging

21
Q

how does ionising radiation damage DNA?

A

Ionising radiation can directly damage DNA -
Double strand breaks (DSBs)
Single strand breaks (SSBs)
Energy from radiation breaks covalent bonds
A loss of DNA bases

Or generate products that are able to damage DNA -
Generate reactive oxygen species (ROS)
Through hydrolysis of water
Reactive particles attack DNA

Very serious effects and difficult to repair

22
Q

what is the average annual ionising radiation exposure?

what are the main sources?

A

2.7 mSv a year

48% of that is from radioactive radon gas from the ground

23
Q

how much ionising radiation do you get from a chest x-ray?

transatlantic flight?
chest CT scan?

A

X-ray ~ 0.014 mSv
TA flight ~ 0.08 mSv
CT scan ~ 6.6 mSv

100g brazil nuts ~ 0.01 mSv

24
Q

Radon gas -

where does it come from, what kind of radiation is it and is it harmful?

A

from the ground

can be alpha, beta or gamma particles

UK average dose is 1.3 mSv

1000 lung cancer deaths naturally attributed to Radon

homes in places like Cornwall need to be protected from it

25
Q

annual limit of radiation?

A

Average = 2.7 millisieverts (mSv) of radiation per year, but the annual limit for nuclear industry employees = 20mSv

Level where white cell changes can be observed = 100mSv

Radiation sickness (lose 50% of white blood cell count) = 1000mSv

26
Q

sources of radiation in research?

A

use radiation a lot in research to cause DNA damage, mutations and understand cancer

32P-ATP, 3H (tritium)

27
Q

what is the initial DNA damage response (DDR)?

A
  1. Sensors - detect that there is some damage (include MRN complex for DSB repair, PARP)
  2. Mediators - next step, initiate the process of repair (BRCA1, RAD17)
  3. Signal transducers - like PIKK kinases such as ATM. basically just a cell calling for help/recruit what is needed to fix the damage
  4. Repair effectors - polymerases, p53, BRCA1 etc… not all for DNA repair, some stop cell cycle for example
28
Q

what are PARPs?

A

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes

DNA damage occurs, PARPs detect and bind to the sites of damage, particularly single-strand breaks.

Upon binding, PARPs add PAR chains to themselves and other target proteins. This causes the recruitment of DNA repair proteins to the damage site, promoting the repair process

29
Q

is DNA repair good?

A

99.999% efficient

30
Q

explain one of the major kinds of damage caused by alkylating agents

A

N-nitroso compounds (NOCs)) can cause guanine (G) to become methylated - alkyl group is transferred to the oxygen atom at the sixth position of guanine

Guanine becomes ‘O-6-Methylguanine’, which pairs with T instead of C

this is one of the major mutagenic lesions caused by alkylating agents

31
Q

alkylating agents can cause guanine to be methylated.

give an exmaple of DNA repair that reverses the chemical damage

A

O6-methylguanine-DNA methyltransferase (MGMT), also known as the “Suicide enzyme”

The alkyl group (CH3) is transferred from guanine to the enzyme itself, inactivating the protein and causing its own degradation

O-6-Methylguanine is back to being guanine and normal base pairing is restored

32
Q

is MGMT involved in cancer?

A

methylation of the promoter for the MGMT gene is found in 40% of cancers

(methylation reduces/prevents transcription)

33
Q

what does ‘base excision repair’ repair?

how does it work?

A

Repairs deamination and abasic sites

  1. During replication, deamination can be recognised by uracil DNA glycosylase. Glycosylase enzymes travel along checking the bases
  2. This enzyme ‘flips’ the deaminated base out, like putting a sticky tab on a page that needs to be reviewed.
    The DNA repair pathway needs to come and fix it
  3. Uracil DNA glycosylase will then ‘excise’/remove the base
  4. AP endonuclease and phosphodiesterase come along and remove the section of sugar-phosphate backbone, leaving a SS break
  5. DNA polymerase adds a new, correct nucleotide, DNA ligase seals the nick
34
Q

what is the nucleotide excision repair pathway used to fix, and how does it work?

A

Or the short patch repair pathway, for when more than one base is involved - pyrimidine dimers

  1. Damage is recognised by large multiprotein complexes scanning the DNA
  2. An excision nuclease cuts/excises the damage site a few bases upstream and downstream of the damage site - just takes out a little chunk
  3. DNA helicase removes this cut out damaged section
  4. The 12-ish nucleotide gap left behind is filled with DNA synthesised by polymerase, with DNA ligase sealing the nicks again
35
Q

what is non-homologous end joining?

A

one of two methods for fixing DSBs, it occurs in G1 of the cell cycle

it simply rejoins the breaks and is therefore error-prone, genetic information is often lost

36
Q

how does NHEJ work?

A
  1. Recognition of DNA Ends: NHEJ is initiated when a cell detects double-strand breaks (DSBs) in the DNA. MRN complex resects the DNA slightly to give a 3’ overhang
  2. Binding of Repair Proteins: Proteins Ku70/Ku80 and DNA PKA, quickly bind to each of the broken DNA ends, forming a complex known as the Ku heterodimer, pulling the two ends together. Note, DNA-PKA phosphorylates itself and then other proteins
  3. End Processing: endonucleases then come along and remove the 3’ overhang
  4. Ligation: DNA ligase joins the ends together
37
Q

what are the benefits and drawbacks of NHEJ?

A

benefits Quick and easy avoiding large scale DDRs, can occur throughout the cell cycle…

No template needed
Drawbacks Error prone - lose some DNA in the resection, can cause insertions and deletions

38
Q

when does homologous recombination occur?

A

Only occurs in S-phase and G2 because it uses the newly synthesised DNA as a template
*It’s complex, but results in error-free repair

39
Q

what are the benefits and limitations of homologous recombination?

A

Benefits - error free/restores correct DNA sequence
Limitations - need a sister chromatid (so only really in G2), not an option for non-dividing cells like stem cells, neurons etc…
Can cause loss of heterozygosity (LOH)

40
Q

how does homologous recombination occur?

A

ATM kinase is what recognises the DS-break. ATM phosphorylates histone 2AX (H2AX) to slightly relax the nucleosome structure. Phosphorylated H2AX is used as a marker for DSBs and DNA damage in labs

At the double stranded break, MRN complex resects the two strands to produce 3’ overhangs (because this is needed for DNA polymerase to later be able to add on)

Proteins like Rad51 and RPA coat the 3’ overhang, causing recruitment of more proteins such as BRCA1 and 2 to help with…
Strand invasion, where the damaged DNA finds its sister chromatid at the section in need of repair, and ‘invades’ so that it can get a look at a correct version of the complementary strand and use it as a template. Note, this first ‘crossover’ where the damaged strand invades is called a holliday junction

DNA polymerase will use the undamaged template to synthesise the missing section of DNA and the once broken strand rejoins its original strand (note, now that there are two crosses over, 1. Where the strand first invaded and now 2. Where it is leaving to rejoin it’s original strand, this is called a double holliday junction)

The newly synthesised DNA now acts as a template itself for the other break - in the other DNA strand (as this is for DS breaks)

DNA ligase seals the nicks

41
Q

homologous recombination - what does ATM kinase do?

A

recognises the break, phosphorylates Histone 2AX to relax nucleosome structure

this means phosphorylated H2AX is a marker for DSBs and DNA damage in the lab

42
Q

what are PIKKs?

A

Phosphatidylinositol 3-kinase-related kinase

These are key components of the DNA repair pathways
They work as switches - phosphorylating proteins to activate them/create binding sites etc…

Phosphatases then typically remove the phosphates to deactivate

43
Q

what are some examples of PIKKs and their roles?

A

ATM (Ataxia-telangiectasia mutated protein) -
Largely responds to classic DS breaks in homologous recombination

ATR: Ataxia telangiectasia and Rad3 related protein) -
More used with SS-DNA, stalled replication forks etc…
phosphorylates several downstream targets to halt the cell cycle, stabilize replication forks, and promote DNA repair pathways

DNA-PK
A DNA protein kinase
Non homologous end joining

44
Q

what are telomeres?

A

specialised structures on the end of chromosomes, protecting them/hiding them

they don’t code for proteins but do code for RNA

the ends of chromosomes look like a DSB and would initiate some kind of DNA damage response if they weren’t hidden

45
Q

how do telomeres evade the DDR pathways?

A

their structure hides the loose end

their associated proteins…

46
Q

explain how the structure of the telomere hides the end of the chromosome

A

two loops, one of the strands loops around and invades itself, looks like a holliday junction - this is the D-loop, (the smaller intratelomeric loop)

the other strand essentially ‘spoons’ this D-loop. this would be the T-loop

47
Q
A

genetics 2 (15 decks)

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