Protecting the Genome

Question 1

Outline the causes of DNA damage

Answer 1

Endogenous Chemical - ROS, Spontaneous hydrolysis and Alkylation.
Exogenous Chemical - Diet (nitrosamines), Pollutants, Drugs (chemo).
Endogenous Physical - Mechanical DNA damage. Exogenous Physical - IR and UV-B.
Endogenous Biological - DNA replication errors, Transposons, Chromosome Missegregration.
Exogenous Biological - Retroviruses

Question 2

Explain how ROS can damage the DNA and where they come from

Answer 2

ROS come from incomplete reduction of oxygen in oxidative phosphorylation. They react with the DNA and causes chemical changes to it

Question 3

Explain the effect of different types of radiation on DNA

Answer 3

DNA absorbs 260nm most efficiently but that’s absorbed by atmosphere. UVB (295-320nm) gets through and can damage cells. It can either directly effect the DNA, or create an OH ion after interaction with water which interacts with DNA. Also, ionising radiation such as X rays and Gamma rays can damage DNA.

Question 4

How can we avoid damage to our genome?

Answer 4

We cannot avoid ionising radiation, but we can avoid tobacco smoke, certain foods in our diet etc. Much endogenous damage is unpreventable and we must deal with it when it happens (repair).

Question 5

Explain the body’s mechanism for neutralising ROS

Answer 5

Superoxide Dismutase enzyme catalyses the breakdown of superoxide (O2 free radicals) into Oxygen and H2O2. Catalase or Glutathione Peroxidase converts H2O2 into H2O and O2. Glutathione Peroxidase does this by oxidising Glutathione to form dimer GSSG which is reduced back to form Glutathione by Glutathione Reductase. High GSSG:GSH ratio indicates high oxidative stress.

Question 6

Explain the body’s mechanism for avoiding DNA replication errors

Answer 6

The DNA Polymerases of the replisome have inbuilt proof reading activity, so during chain elongation they can sense misincorporation, will delete a few nucleotides using its 3’-5’ exonuclease activity, and start again.

Question 7

Explain the body’s mechanism for avoiding chromosome segregation errors

Answer 7

The Spindle Checkpoint Assembly prevents abberant segregation. Specialised proteins in the kinetochore (centromeric protein complex) sense spindle tension - a low level will indicate that the spindles are not properly attached to the centromere, and a signal is sent which inactivates the APC/C. The APC/C triggers anaphase by the degredation of cohesin.

Question 8

Describe the cause, consequence, and repair method of base modification/loss

Answer 8

Cause: Oxidation, Hydrolysis, Alyklation
Consequences: Point mutation, replication stalling
Repair: Base excision repair

Question 9

Describe the different types of base modification

Answer 9

Oxidation of Guanine to 8-OxoGuanine by ROS, 8-OG will bond with A if not repaired
Depurination - Hydrolysis of G, leading to an abasic site which blocks replication and transcription
Hydrolytic deamination of C to U - U in DNA blocks replication
Hydrolytic deamination of 5-mC to T - Becomes a fixed mutation in DNA

Question 10

Describe the cause, consequence, and repair method of single strand breaks

Answer 10

Cause: ROS, IR
Consequences: Converted to DSB by replication
Repair: HR or NHEJ (when DSB), BER

Question 11

Describe the cause, consequence, and repair method of helix distorting damage like thymine dimers or Intra-strand links

Answer 11

Cause: UV light (T-dimers), Bifunctional alkylating agents (intralinked strands)
Consequences: Replication stalling
Repair: Nucleotide excision repair

Question 12

Describe the cause, consequence, and repair method of mismatches and IDLs

Answer 12

Cause: Replication errors (frequency of 1/10^8) such as replication slippage or nucleotide misincorporation
Consequences: Replication stalling
Repair: Mismatch repair

Question 13

Describe the cause, consequence, and repair method of Inter-strand links

Answer 13

Cause: Bifunctional alkylating agents
Consequence: Replication stalling, Cell death
Repair: Homologous Repair

Question 14

Describe the cause, consequence, and repair method of DSBs

Answer 14

Cause: Replicated SSBs, ROS, IR, Mechanical Breaks
Consequences: Chromosomal deletions, inversions, translocations
Repair; NHEJ and HR (When sister chromatid available)

Question 15

Describe the process of Base Excision Repair

Answer 15

DNA glycosylases (variable to detect different base damage) identify and remove the damaged base by cleaving the base-backbone bond to leave an AP site
A 5’ AP endonuclease cuts the sugar phosphate backbone
The baseless sugar phosphate is removed by dRase
The gap is filled and sealed by DNA Polymerase and Ligase, using the undamaged strand as a template
These last 2 steps contribute to PARP recognised ssb repair

Question 16

Describe the process of Nucleotide Excision Repair

Answer 16

There are 2 pathways for NER, if the damage is in a region transcribed by RNA Pol II it’s Transcription Coupled Pathway, if not it’s Global Pathway. In the Global pathway, XPC recognises the damage, in TCR it’s RNA Pol II.
The DNA is unwound around the damage by helicases XPB and XPD.
XPF and XPG endonucleases make incisions a few nucleotides upstream and downstream of the damage.
25-30 nucleotides are removed, and DNA Polymerase and DNA Ligase fill and seal

Question 17

Describe some diseases associated with mutations in proteins involved in NER

Answer 17

Xeroderma Pigmentosum is an autosomal recessive disease which is characterised by mutations in XP protein genes which causes UV light sensitivity, leading to corneal ulcers and skin dryness. Cockayne’s Syndrome is an autosomal recessive disease associate with mutations in CSA or CSB proteins which are important for post transcriptional modifications of RNA Pol II. Symptoms include short stature and neurological dysfunction and mild skin sensitivity.

Question 18

Describe the process of Mismatch Repair

Answer 18

MutSa or MutLa recognises the mismatch/IDL.
They diffuse away from the mismatch until they reach nicks in the newly synthesised strand (this is how they differentiate new strands from parental) bound by PCNA (DNA Polymerase clamp) or RCF (loading factor).
EXO1 (exonuclease) degrades the new strand towards and through the mismatch region.
PCNA recruits DNA Pol to fill, and DNA Ligase ligates.
Hereditary Nonpolyposis Colorectal Cancer (5-7% all colon cancers) is an autosomal dominant disease which arises from mutations in any MMR related genes like MutS or MutL

Question 19

Describe the process of Homologous Repair

Answer 19

After a dsb occurs, exonucleases bind to the ends of the exposed DNA and cleave nucleotides to give ss overhangs
RPA protects the SS overhangs
BRCA1 and BRCA2 help RAD51 to bind to the DNA, replacing RPA
The DNA end with RAD51 invades the homologous DNA, causing a Holliday Junction, and DNA Polymerase repairs the strand by using the other strand as a template
More proteins (like BLM) remove the HJ so that the two strands can seperate. DNA Polymerase and Ligase

Question 20

In which other scenarios is HR important?

Answer 20

In meiotic division where it introduces diversity by crossing over

Question 21

What happens when key HR proteins are mutated and how does it relate to cancer?

Answer 21

When key HR proteins like BRCA 1 and 2 have mutations, there is a decreased viability of cultured human cells. Also, mutations in BRCA1 and 2 genes are a characteristic of some cancers

Question 22

What is a limitation of the fact that HR uses a template and how does the body overcome this?

Answer 22

If HR uses an inappropriate sequence as a template, such as a repeat region, it is genome destabilising. The body minimises this risk by inactivating the HR pathway when there is no sister chromatid present to act as a template - G1

Question 23

Describe the process of Non-Homologous End Joining

Answer 23

Ku70/80 heterodimers bind to each end of the damaged DNA.
If the ends are blunt and undamaged they can be ligated immediately
Ku recruits the catalytic subunit of DNA protein kinase to form an active DNA-PK complex
DNA-PK recruits and activates Artemis exonuclease to trim the DNA ends and DNA Polymerase to add nucleotides
DNA Ligase is recruited to seal nicks
The end result of NHEJ is either a repaired region or an indel

Question 24

What other applications does NHEJ have aside from repairing DSBs?

Answer 24

Used in Immunoglobulin rearrangement and T cell receptor rearrangment so deficiencies or mutations in NHEJ proteins can lead to immunodeficiency

Question 25

Explain the DNA damage response

Answer 25

Aswell as repair, the response includes the induction of transcription, cell cycle arrest, and apoptosis

Question 26

Detail the rang of proteins involved in the DNA damage response

Answer 26

Sensor proteins detect the DNA damage and along with mediators, recruit and activate transducer kinases ATM and ATR. Positive feedback between the mediators and transducers leads to the maintenance and amplification of the signal.
Transducer kinases phosphorylate effector kinase Chk1 and 2, which phosphorylate affector proteins which control various TFs, apoptosis, and cell cycle arrest. Which response is induced depends on the severity of the DNA damage and the strength and duration of the kinase response and phosphorylations

Question 27

How does p53 act in terms of the DNA damage response?

Answer 27

The tumour supressor and TF p53 may act in transcription-dependent or -independent ways to promote reversible cell cycle arrest or apoptosis

Question 28

How do Cdc25 proteins act in terms of cell cycle arrest

Answer 28

Cdc25 proteins are Chk1/2 activated are phosphatases that down-regulate Cdk activity to arrest cell cycle progression

Question 29

Why is it advantageous to arrest cell cycle in response to DNA damage?

Answer 29

Cell cycle arrest allows DNA damage to be repaired before DNA is replicated (G1/S checkpoint) or before it’s transmitted to daughter cells (G2/M checkpoint). It avoids complications such as stalling at the DNA replisome

Question 30

What is the advantage of a cell undergoing apoptosis in response to DNA damage?

Answer 30

Apoptosis eliminates the high risk of tumorigenesis from cells where damage was too severe to be properly repaired

Question 31

How can IR and chemotherapy be used to treat cancer?

Answer 31

IR and chemo cause DSBs. Because cancer cells grow and divide more frequently than normal cells, they are more susceptible to such agents.

Question 32

What are side effects of IR and chemotherapy?

Answer 32

These treatments have many side effects if they interact with normal cells. They may damage cells in the gi tract, bone marrow, and hair follicle. This leads to nausea, immunodeficiency and hair loss. Also, if the DNA of normal cells is mutated enough it can lead to cancer

Question 33

Explain the concept of synthetic lethality

Answer 33

This is a treatment technique used for cancers with known defects in the DNA repair genes. It involves using PARP inhibitors to treat the cancer without harming normal cells

Question 34

What is the effect of PARP inhibition on DNA repair?

Answer 34

It impairs BER when repairing ssb

Question 35

What happens to ssbs when unrepaired?

Answer 35

When replicated, they become DSBs

Question 36

What is the effect of PARP inhibitors on normal and BRCA deficient cells?

Answer 36

DSBs are repaired by normal cells using HR, but BRCA deficient cells have to use BER, which relies on PARP. The PARP is inhibited, which means that the DSBs are not repaired and the cell dies