Lecture 12 - DNA repair & maintaining stability

Question 1

Why do multiple DNA repair pathways exist in cells?

Answer 1

• recognise DNA damage has occurred
• recruit repair proteins to the site of damage
• repair damaged DNA & restore function
• OR force cells to undergo apoptosis
• DNA repair is 99.999% efficient

Question 2

What makes up the DNA damage response (DDR)?

Answer 2

First job is to stop the cell cycle

• DNA damage Sensors (MRN complex, PARP)
• Mediators (BRCA1, RAD1)
• Signals transducers (PIKK kinases - ATM, ATR etc.)
• Repair effectors (Chk1, BRCA1. p53, polymerases)

Question 3

How does the DNA damage response work?

Answer 3

• Reverse the chemical reaction that caused DNA damage
• Directly remove damaged DNA & replace it with new - correct DNA

Question 4

How does direct chemical reversal of damage occur?

Answer 4

• alkylating agents (N-nitroso compounds (NOCs))
• can cause guanine (G) to become methylated
• Guanine > O-6 methylguanine
• Pairs with T instead of C
• One of the major mutagenic lesions caused by alkylating agents

Question 5

How are these lesions repaired?

Answer 5

• O6-methylguanine-DNA methyltransferase (MGMT)
• “suicide enzyme”
• The alkyl group (CH3) is transferred from guanine to the enzyme itself
• Inactivates the protein & causes its own degradation
• O-6-Methylguanine > guanine and normal base pairing is restored
• Methylation of the promoter for this gene is found in 40% of cancers

Methylation turns a gene off when bound to a promoter

Question 6

What is Base excision repair (BER)?

Answer 6

• can be long or short patch repair
• determined by the type of lesion, cell cycle stage & other factors

Question 7

What are the key proteins involved in short patch repair?

Answer 7

• Glycosylases
• AP endonuclease
• Repair enzymes (Polymerase/ligase)

Question 8

What do glycosylases do?

Answer 8

• Travel along DNA & check bases (excises abnormal bases)
• Creates an apurinic/apyramidinic (AP) site - No base present

Question 9

What does AP endonuclease do?

Answer 9

Cleaves the DNA - creates a ‘gap’

Question 10

What do repair enzymes (polymerase/ligase) do?

Answer 10

Fil the gap with the correct sequence

Question 11

What is Nucleotide excision repair (NER)?

Answer 11

• repairs bulky lesions e.g. thymine dimers
• rather than single base errors

Question 12

What are the key proteins involved in NER?

Answer 12

• Large multiprotein complex scans DNA
• Looking for distortions in the structure
• Cleaves DNA either side of distortion
• DNA helicase displaces the fragment
• Polymerases & ligases fill the gap
• Restores the correct sequence

Question 13

How are DNA double strand break repair occur?

Answer 13

• Non-homologous end joining
• Homology Directed Repair

Stops the cell & drives apoptosis if it can’t be repaired.

Question 14

What are PIKKs?

Answer 14

Phosphatidylinositol 3-kinase-related kinase (PIKKs) are key components of the DRR:
- protein kinases
- phosphorylate other proteins
- creating binding sites
- or activates enzymes

Question 15

What are cellular responses to the DNA damage response?

Answer 15

• Replisome stability
• Transcription
• Cell cycle
• Energy/Autophagy
• Chromatin remodelling
• Repair
• RNA processing
• Apoptosis

Question 16

What is non-homologous end joining (NHEJ)?

Answer 16

Predominant method of DSB repair in humans
- ends are “simply” joined together
- involves DNA-PK & Ku proteins
- DNA-PK phosphorylates itself & then other proteins
- DNA ends are resected (cut)
- Then joined together by DNA ligases

Question 17

What are the benefits of NHEJ?

Answer 17

• Quickly repairs breaks
• Can occur throughout the cell cycle
• No template needed for repair
• Then joined together by DNA ligases

Question 18

What are the limitations of NHEJ?

Answer 18

• can cause insertions/deletions
• error-prone (mutations)
• 2000 genomic “scars” per cell by 70yrs old

Question 19

What is Homology Directed Repair (HDR)?

Answer 19

A specialised method of DSB repair
- accurate DNA repair using a template
- ATM kinase recognises the DSB
- H2AX is phosphorylated (YG2AX)
- Is a marker of DSBs in cells
- Recruits other HR factors
- Rad51 facilitates strand invasion
- Polymerases uses template to create new DNA

Repair occurs using template. Add phosphate group to histone proteins to draw attention to this chromosome

Question 20

What are the benefits of HDR?

Answer 20

Restores correct DNA sequence

Question 21

What are the limitations of HDR?

Answer 21

• requires a sister chromatid
• can only occur at specific times in cell cycle
• not an option for non-dividing cells (neurons)
• can cause loss of heterozygosity (LOH)

Question 22

What does CRISPR use?

Answer 22

homologous recombination

Question 23

What can reduce genomic instability?

Answer 23

Structural properties of DNA

Question 24

What happens at the end of DNA?

Answer 24

Double strand break found at the end. This activates DNA damage response pathways.

Cell cycle - arrest:
- ATM kinase
- ATR kinase

DNA repair:
- HDR
- NHEJ

Protected from DNA damage response pathways

Question 25

What is involved in DSBs?

Answer 25

ATM + DNA-PK

Question 26

What is involved in SSBs?

Answer 26

ATR

Question 27

What are telomeres?

Answer 27

Telomeres found at the end of DNA & hides the DSB - specialised structures that protect the end of chromosomes - TTAGG repetitive sequences - Non-protein coding - Would initiate a DDR if left unprotected - The "End Protection Problem"

Question 28

How do Telomeres evade the DDR?

Answer 28

- Telomere structure - Associated proteins - Telomerase extension (in some cells)

Question 29

Describe Telomere structure

Answer 29

2 key DNA structures are present in a telomere - T-loop (main loop) - D-loop (small loop) These hide the DNA end. D-loop resembles homologous repair

Question 30

Describe Telomere structure (advanced)

Answer 30

- Telomeric DNA is also supported by proteins - The Shelterin complex - Interacts with telomeric DNA & performs many roles. - Shelterin proteins help the telomere - Reinforce these structures - Hide the DNA end TPP1/POT1 - suppress ATR TRF2 - suppresses ATM - prevents a DDR - Thought to interact with ATM - Block its MRN induced activation - By preventing MRN from recognising the DNA end.

Question 31

What is Telomere shortening

Answer 31

The Hayflick limit Forward & Backward synthesis creates gaps. Replication forks fill in gaps. Not found in telomeres which mean that when telomere DNA is replicated, an overhand is left & is cut off, meaning some telomeric DNA is removed. This is okay till the telomeres get really short & double strand break is exposed, leading to initiation of DNA damage repair mechanisms. This leads to ageing. The Hayflick limit is the number of times the cell can divide before the loss of telomeres.

Question 32

What is the Hayflick limit?

Answer 32

The number of times a cell population will divide before cell division stops (40-60 divisions). Outcomes: Senescence - Crisis - Apoptosis - Adaption

Question 33

What is senescence?

Answer 33

When a cell stops dividing permanently - go into G0 not cell cycle. The damage will accumulate & apoptosis will be initiated. Embryonic cells keep their telomeres long, as well as stem cells. This is because they last a long time & divide a large number of times. In most somatic cells, telomeres get progressively shorter. The crisis point is reached & apoptosis occurs. In cancer cells, replication occurs despite loss of telomeres - through re-activation of telomerase.

Question 34

What is telomerase?

Answer 34

- an enzyme - synthesises more TTAGGG repeats to extend short telomeres - formed an RNA component & catalytic reverse transcriptase enzyme - not expressed or active in most tissues - is active in stem cells - some immune cells (B-cells) - germ cells - cancer cells (reactivated) Mutations (C>T in the hTERT promoter can upregulate telomerase in cancer Anti-telomerase treatments for cancer RNA template & telomerase enzyme. Isn't exposed in all tissues as, don't want skin cells that has been exposed to carcinogens to last forever. Is found in gametes.