GEN 8: Protecting the Genome Flashcards
1
Q
Observe the learning outcomes of this session
A
2
Q
What are the two basic sources of DNA damage?
A
- reactive chemicals:
- free radicals from our ‘basic metabolism’
- radiation
3
Q
What are free radicals and how do they damage DNA?
A
- they are molecules with an unpaired electrons
- The most common types in our cells are reactive oxygen species (ROS) generated most often by the incomplete reduction of oxygen during mitochondrial oxidative phosphorylation.
- ROS oxidise the DNA
4
Q
What are the forms of chemical endogenous DNA damage sources?
A
- ROS (oxidation)
- spontaneous hydrolysis
- alkylating agents
5
Q
Which UV rays are most damaging?
A
- DNA absorbs UV light most efficiently at 260nm wavelength.
- This, and shorter wavelengths (higher energy), are highly damaging to DNA but fortunately these are efficiently absorbed by the atmosphere.
- Wavelengths in the range 295-320nm (termed UVB) do reach us, however, and damage DNA in the skin cells that absorb them.
6
Q
What are some other types of radiation that aren’t UV?
A
- X-rays
- radioactive elements (natural or man-made)
7
Q
What is ionizing radiation (IR)?
A
- penetrating radiation that causes DNA damage is called ionizing radiation (IR)
- It includes both electromagnetic waves (e.g. gamma-rays, X-rays, UVC rays) and atomic or subatomic particles with sufficient energy to dislodge electrons from the atoms with which they collide.
- Most of the IR we experience is natural, such as cosmic radiation and unstable isotopes in rocks (e.g. Uranium-235).
- Much less is manmade (e.g. hospital X-ray equipment).
8
Q
How does IR damage DNA?
A
- either directly or by generating ROS from water
9
Q
What type of DNA damage are ionising and UVB radiation?
A
- exogenous
- physical
10
Q
What other exogenous chemicals cause DNA damage?
A
- Environmental pollutants (e.g. alkylating agents and other chemicals in car fumes, tobacco smoke, crop sprays etc).
- Natural toxins (e.g. fungal aflotoxins[SAM1]).
- Dietary chemicals (e.g. products of cooking or curing processes including nitrosamines)
- Anti-cancer drugs (e.g. cisplatin)
11
Q
What is a form of endogenous physical damage?
A
- Though less obvious, mechanical damage can arise from errors in chromosome replication or segregation, causing chromosomes to be torn apart by the mitotic spindle apparatus.
- For example, this can result from unscheduled replication of centromeric DNA, as illustrated in the next image.
12
Q
Explain how mechanical DNA damage occurs
A
- The blobs and rings represent centromeres and cohesin, respectively.
- Thin lines represent spindles attached to centromeres at metaphase (centre) and anaphase (right) and pulling chromatids to opposite poles to the left or right.
- Failure of controlled replication origin firing (see GEN6) may cause re-replication in the centromeric region resulting in one sister with two centromeres.
- Depending on how the spindles attach, the outcome may be chromosome mis-segregation (top), or physical breakage of the chromosomal DNA (bottom).
13
Q
Give examples of biological genome damage sources
A
- viruses
- transposons
- DNA replication errors
- chromosome segregation failures
14
Q
Describe how viruses are a biological DNA damage source
A
- Many viruses (e.g. retroviruses) insert their genomes into the genome of their host cell.
15
Q
Describe how transposons are a biological DNA damage source
A
- though rare in human cells, transposons can move from site to site within a genome causing insertional or excisional mutagenesis.
16
Q
Describe how DNA replication errors are a biological DNA damage source
A
- DNA replication errors are another source of biological DNA damage. These result from:
- Nucleotide misincorporation by DNA Polymerase
- Replication slippage during microsatellite DNA replication. The image illustrates slippage during replication of a stretch of DNA with a single nucleotide repeat (AAAA). Note how insertion-deletion loops (IDLs) of unpaired bases are formed.
17
Q
Describe how chromosome segregation failures are a biological source of DNA damage
A
- Two pairs of sister chromatids are depicted at metaphase (left) and the ensuing anaphase (right).
- The top pair segregates normally, each being pulled to opposite poles of the cell by spindles (microtubules) attached to centromeric proteins (kinetochores).
- Each daughter cell will therefore receive one copy of the top chromosome.
- The bottom pair, however, has one sister that fails to attach to the spindle.
- As a result, both sisters will move to a single pole, so one daughter cell will receive two copies and the other will receive none.
18
Q
Complete the table of the sources of DNA damage
A
19
Q
What are the main types of DNA damage?
A
- base modification or loss
- single strand breaks
- bulky adducts:
- intra-strand-X-links
- CPDs
- mismatches and IDLs
- inter-strand X-links
- double strand breaks
20
Q
Describe the causes, consequences and repair pathways of base modifications or losses
A
- causes:
- oxidation (ROS/IR)
- hydrolysis
- alkylation
- consequences:
- point mutations
- replication stalling
- repair pathways:
- base excision repair
- direct repair
21
Q
Describe the causes, consequences and repair pathways of SSB (single strand breaks)
A
- causes:
- ROS
- IR
- consequences:
- converted to double strand breaks by DNA replication
- repair pathway:
- base excision repair
22
Q
Describe the causes, consequences and repair pathways of bulky adducts and intrastrand crosslinks
and additional info
A
- causes: UVB or alkylating agents
- consequences: replication stalling
- repair pathway: nucleotide excision repair
- many cancer chemotherapies use cross-linking agents
23
Q
Describe the causes, frequency, consequences and repair pathways of mismatches and IDLs
A
- causes: replication errors
- frequency: after proofreading the mammalian replisome generate only one mismatch per 108 nucleotides
- consequences: replication stalling
- repair pathway: mismatch repair (MMR)
24
Q
Describe the causes, consequences and repair pathways of inter-strand crosslinks
- uses
A
- causes: bi-functional alkylating agents
- consequences: replication stalling, cell death
- repair pathway: HR
- many cancer chemotherapies use crosslinking agents
25
Q
Describe the causes, consequences and repair pathways of double-strand breaks
A
- causes: ROS, IR, mechanical breaks, replications of SSB
- consequences: if unrepaired may lead to small or large mutations including chromosomal deletions, inversions, translocations and chromosome loss
- repair pathway: non homologous end joining (NHEJ), homologous-directed repair (HR)
26
Q
Describe the oxidation of guanine to 8-oxoguanine (base modification)
A
- A. The base guanine can be oxidised by ROS to form 8-oxoguanine (8-OG), but this can be repaired by base excision repair.
- B. If not repaired, 8-OG (boxed) pairs with A instead of C during DNA replication.
27
Q
Describe depurination: hydrolysis of guanine to form an abasic site (base modification)
A
- Depurination is the removal of a purine base (Guanine or Adenine) from DNA by hydrolysis, leaving an abasic site (i.e. there is no base there).
- This blocks replication and transcription and needs repairing by base excision repair.
28
Q
Describe the hydrolytic deamination of cytosine to uracil (base modification)
A
- Deamination is the removal of an amino group. If this happens to cytosine, it becomes uracil.
- Uracil in DNA is thought to block DNA replication and must be repaired by base excision repair.
29
Q
Describe the deamination of 5-methyl-cytosine to thymine (base modification)
A
- If 5-methyl-cytosine is deaminated, it becomes thymine which, unless removed, becomes a fixed mutation in the DNA sequence.
30
Q
What are three mechanisms our cells have to minimise some types of endogenous DNA damage?
A
- neutralising reactive oxygen species (ROS)
- avoiding DNA replication errors
- avoiding chromosome segregation errors
31
Q
Describe how reactive oxygen species (ROS) are neutralised
A
- Naturally occurring antioxidants such vitamin C, beta-carotene and glutathione, provide some protection from ROS, but further protection is provided enzymatically.
- As indicated in the following image, superoxide dismutase (SOD) catalyses the decomposition of superoxide radicals into hydrogen peroxide, which is then converted into water by catalase (CAT) or glutathione peroxidase (GPx).
- In the latter reaction, the tripeptide glutathione (GSH) is oxidised to form a dimer (GSSG) which must then be reduced back to GSH by glutathione reductase (GH).
32
Q
Describe how DNA replication errors are avoided
A
- The DNA polymerases of the replisome (DNA pol α, 𝛿 and ε) have inbuilt proof-reading activity.
- During chain elongation they can sense nucleotide mis-incorporation and use their 3’-to-5’ exonuclease activity to remove the offending nucleotide before resuming synthesis, as illustrated in the next image.
33
Q
Describe how chromosome segregation errors are avoided
A
- A mechanism called the Spindle Assembly Checkpoint (SAC) prevents aberrant chromosome segregation at anaphase and any resulting aneuploidy or chromosome breaks.
- riefly, the SAC uses specialised proteins to sense the spindle tension. When this is too low because of incorrect attachment of spindles, a signal is sent that keeps APC/C in an inactive state.
- Recall from GEN6 that APC/C is responsible for triggering anaphase by promoting the degradation of cohesin, the protein that holds sister chromatids together
- In this way anaphase can only proceed when all chromosomes are correctly attached to spindles. The SAC mechanism is outlined on the following image (click to enlarge).
34
Q
What are the DNA repair pathways?
A
- mismatch repair (MMR)
- Base Excision Repair (BER)
- Nucleotide Excision Repair (NER)
- Homologous Recombination (HR)
- Non-Homologous End-Joining (NHEJ)
35
Q
Place the correct pathway in the blue boxes
A
36
Q
Describe base excision repair
A
- Base excision repair is required to remove different types of base damage.
- This could be a removing uracil, 8-oxo-guanine (as shown here) or dealing with missing bases.
- Four steps are involved in this process.
- The key enzyme involved in this repair mechanism is DNA glycosylase.
- DNA gyclosylase identifies the modified base and removes it from the double helix.
- This results in an empty sugar phosphate region.
- An endonuclease then cuts the DNA backbone.
- The baseless sugar-phosphate is removed and DNA polymerase adds a new base, and DNA ligase seals the gap.
37
Q
Describe nucleotide excision repair (NER)
A
- Nucleotide excision repair removes bulky damage such a pyrimidine dimers.
- You can see here that these two Ts represent a pyrimidine dimer, the type of damage caused by UV radiation.
- This type of damage will be recognised by RNA pol II, if it occurs in transcribed DNA.
- However, if it occurs elsewhere in the genome, the protein XPC recognises it.
- Multiple proteins are involved in DNA repair and you will see many starting with XP, after the disease Xeroderma pigmentosum (XP).
- This is an autosomal recessive disease caused by mutations in an XP gene.
- XP patients suffer from sensitivity to UV light resulting in corneal ulcerations and dry skin prone to blistering and cancer.
- DNA around the damage is unwound by helicases.
- Specifically, proteins XPD and XPB which are helicases. Endonucleases, in this case, XPF and XPG, snip the DNA up and downstream and 25-30 nucleotides are excised. \
- New DNA is synthesised by DNA polymerase, and then sealed by DNA ligase.
38
Q
Describe mismatch repair (MMR)
A
- So DNA is replicated and proofreading ensures most errors are rectified. However, this is not 100% efficient.
- As such, we can be left with mismatched bases.
- Mismatch repair can tackle this in a similar way to excision repair, by cutting out the damaged DNA and synthesising a new strand. However, in this case, there is no recognisable damage.
- The bases are simply incorrect.
- In this figure you can see guanine, which should pair with cytosine, is actually paired with thymine due to a replication error.
- When new DNA is synthesised, it contains nicks, that you can see here.
- This distinguishes the daughter strand, contained the mismatch, from the parent strand, and is therefore a way the cell can determine which strand has the error.
- The protein MutS binds to the mismatched DNA and forms a complex with MutL.
- These then slide along the daughter strand until they find one of these nicks.
- An exonuclease degrades the mismatched region of DNA.
- This gap is then filled once again by DNA polymerase in the 5’ to 3’ direction, and then sealed by DNA ligase.
- The importance of the mismatch repair pathway is illustrated by Hereditary Nonpolyposis Colorectal Cancer (HNPCC), which accounts for 5-7% of colon cancers.
HNPCC is inherited in an autosomal dominant fashion as a result of mutations in any of several genes encoding MMR proteins, such as MutSα and MutLα.
39
Q
Why is BER significant?
A
- base excision repair is very versatile because it can choose from multiple glycosylase enzymes, each recognising a different kind of damaged base.
- Furthermore, the last two steps in BER contribute to the repair of IR-induced SSBs that have been recognised by a protein call polyADP ribose polymerase (PARP).
- As we will see later, PARP turns out be an important therapeutic target.
40
Q
Recall the enzymes used in base excision repair
A
- glycosylase
- Endonuclease
- DNA polymerase & ligase
41
Q
Recall the enzymes used in nucleotide excision repair
A
- XPC or RNApolII
- Helicase (XPB, XPD)
- Endonuclease (XPF, XPG)
- DNA polymerase & ligase
42
Q
What are the enzymes used in mismatch repair?
A
- MutL & MutS
- Exonuclease
- DNA polymerase & ligase
43
Q
What processes repair double-stranded DNA damage?
A
- homologous recombination (HR)
- non-homologous end joining (NHEJ).
44
Q
Describe homologous recombination (HR) in more detail
A
- Following a double-strand break in the DNA, exonuclease proteins bind to both broken ends and cleave to produce single stranded overhangs.
- These overhangs are protected by the protein RPA.
- RPA is then replaced by important repair proteins you have have heard of before: BRCA1, BRCA2 and RAD51.
- The end bound by RAD51 then invades the homologous region of the sister chromatid and this forms a Holliday junction.
- Other proteins are involved in this process, however it is just key for you to know here that DNA polymerase synthesises a new strand from this template, and DNA ligase seals the DNA.
- You may have heard of BRCA1 and 2 gene mutations being linked to inherited or acquired cancers, which shows the importance of HR in repairing DNA.
45
Q
Describe non-homologous end joining (NHEJ) in more detail
A
- NHEJ is an error prone process for repairing double-stranded DNA breaks.
- When a break occurs, if the ends are blunt, DNA ligase can seal the gap right away.
- However, if the ends are not blunt the need processing first. Protein Ku70/80 binds to both ends and recruits DNA protein kinase.
- DNA-PK then recruits two more enzymes; an exonuclease called Artemis, and DNA polymerase.
- These can trim and fill in the ends of the broken DNA respectively, creating blunt ends.
- DNA ligase can then be recruited to seal the gap.
- There are aberrant translocations of ends, derived from different chromosomes, associated with cancer.
46
Q
Which DNA repair pathways require which enzymes?
A
47
Q
Choose one or more pathways for each of the descriptions below
A
48
Q
Look at some of the proteins that are involved in the DNA damage response (DDR)
Describe their functions
A
- includes:
- sensors: detect DNA damage
- mediators: recruit and activate transducers
- signal transducing kinases: phosphorylate and activate effector proteins
- effector kinases, effector proteins: lead to transcription, apoptosis and cell cycle arrest
49
Q
Why do you think it might be advantageous to arrest the cell cycle in response to DNA damage?
A
- Cell cycle arrest allows DNA damage to be repaired before the DNA is replicated (G1/S checkpoint) or transmitted to daughter cells (G2/M checkpoint).
- It also avoids complications such as stalling of DNA replisome at damaged DNA.
50
Q
What is the advantage of a cell undergoing apoptosis in response to DNA damage?
A
- Apoptosis eliminates the high risk of tumorigenesis from cells where damage was too severe to be properly repaired.
51
Q
What is the paradox of cancer therapy?
A
- Paradoxically, despite its cancer-causing effects, DNA damage can be used for cancer therapy.
52
Q
Describe radiotherapy and chemotherapy
A
- In the treatment of many cancers, radiotherapy (ionising radiation) and chemotherapeutic drugs work by inducing DNA damage, particularly double strand breaks.
- Because cancer cells grow and divide more frequently that normal cells, they are more susceptible to such agents.
- These treatments have many side effects, such as killing the normally fast-growing cells in the bone marrow, digestive tract and hair follicles, leading to infections, sickness and hair loss.
- More seriously, DNA damage in normal cells increases the risk of developing treatment-related cancers.
- Chemotherapy for cancer is also hampered by cancers that develop resistance to the drug.
53
Q
Describe synthetic lethality
A
- Synthetic lethality is relative new approach for targeting cancers with a known defect in DNA repair, such as a mutation in a BRCA gene. To treat such cancers without harming normal cells, drugs have been developed that inhibit PARP.
- The drug Olaparib is a PARP inhibitor used to treat BRCA1 or BRCA2-deficient ovarian cancers. Other synthetic lethal treatments are under development.
54
Q
Can you predict the effect of a PARP inhibitor on DNA repair?
A
- It will impair the ability of BER to repair single strand breaks.
55
Q
What happens to unrepaired SSBs?
A
- They are converted into DSBs during DNA replication.
56
Q
So what will be the effect of PARP inhibition on normal cells and on BRCA1-deficient cancer cells?
A
- Normal cells will be able to repair the DSB by HR, but the cancer cells will not because BRCA1 is required for DSBR by HR.
- Normal cells will therefore survive while the cancer cells will prone to undergo apoptosis.
- This is summarised in the figure attached showing the mechanism of cell death from synthetic lethality as induced by inhibition of Poly(Adenosine Diphosphate [ADP]–Ribose) Polymerase 1 (PARP1).
