translational W4 - DNA damage & repair

mechanism of endogenous DNA damage

• Damage from within the cell

• Internal biochemical processes generate harmful by-products

mechanism of exogenous DNA damage

• Damage from outside the cell

• Environmental agents that directly interact with DNA

Endogenous Stressors Examples

by-products of cellular reactions interact with DNA

• Cellular respiration (ROS) from metabolism (especially from mitochondria)

• Lipid peroxidation → creates reactive aldehydes that attack DNA

• Spontaneous hydrolysis → can break DNA bonds

• Alkylation by endogenous methylating enzymes'

Errors during DNA replication

Exogenous Stressors Examples

Damage caused by environmental exposures:

1. non-ionising radiation - UV radiation (UV-A, UV-B) → forms pyrimidine dimers

2. Ionising radiation (X-rays, gamma rays) → causes DNA breaks

3. Chemicals:

   • Cigarette smoke, pollution (contain hydrocarbons and alkylating agents)

   • Aflatoxins (from mould) or other plant/microbial toxins

   • Chemotherapy drugs (designed to damage DNA in cancer cells)

4. Diet/lifestyle:

   • Alcohol consumption

   • Polyunsaturated fats (can undergo oxidation)

DNA damage → mutation

• DNA damage is the first step.

• If repair fails, the damage becomes a permanent mutation.

• This mutation can be inherited by daughter cells.

DNA damage VS DNA mutation

• DAMAGE = reversible, physical or chemical alteration of DNA — Recognised and usually repaired by DNA repair enzymes

• MUTATION = irreversible, permanent change in the DNA sequence — Once both strands contain the mutation, not repairable

DNA Damage effect

Can block replication or transcription

DNA mutation effect

• May be silent, harmful, or lead to disease or cancer

• Some mutations add genetic variation (e.g. SNPs – single nucleotide polymorphisms)

what are SNP’s?

• mutations that occur in original sequence over some time

• may not have any significant effects or outcomes on specific individuals

• change that occurs in small portion of population

what are the two types of DNA mutations

1. base substitutions (silent, nonsense, missense)

2. point mutations (transition, transversion)

transition VS transversion point mutations

• transition – pyrimidine to pyrimidine OR purine to purine

• transversion – pyrimidine to purine OR purine to pyrimidine 

which bases are pyrimidines?

C, T, U → 1 carbon nitrogen ring base

which bases are purines?

A + G → 2 carbon nitrogen ring base

what is a silent base substitution?

change in third position of codon, no effect on functional protein

what is a nonsense base substitution?

inserts stop codon, has a non-functional effect - results in truncated protein

what is a missense base substitution?

amino acid change, may change functional protein or may have minimal effects

what is an indel? what is the effect?

• what: deletions or insertions within coding region

• effect: causes a frameshift mutation – alters translational reading frame

  • variety of possible outcomes → may cause dysfunctional disease

what are DNA translocations and what is the effect?

• what: rearrangements between non-homologous regions of chromosomes (e.g. swapping of arms)

• outcome: rearrangement of sequence → i.e. promoter shifted = changes regulation of genes by changing their conditions relative other promoter which can have significant functional effects

  • gene / protein fusions

  • inactivation

  • Overexpression

what causes DNA translocations?

DNA double strand breaks

what are the five major DNA repair mechanisms Mammalian cells use;

1. base excision repair (BER)

2. mismatch repair (MMR)

3. nucleotide excision repair (NER)

4. double-strand break repair

   1. homologous recombination (HR)

   2. non-homologous end joining (NHEJ)

1. base excision repair (BER)

2. mismatch repair (MMR)

3. nucleotide excision repair (NER)

4. double-strand break repair

   1. homologous recombination (HR)

   2. non-homologous end joining (NHEJ)

What are the 4 classes of DNA repair?

1. direct repair

2. excision repair (MMR, BER, NER)

3. double-stranded break repair (HR, NHEJ)

4. interstrand crosslink repair

endonucleases function

creation of nicks in DNA to allow access to and removal of damaged region (cleave phosphodiester bond in DNA)

exonucleases function

cleavage of DNA and nucleotide removal (cleave phosphodiester bond at end of DNA)

DNA helicases function

DNA unwinding to allow access of other proteins

DNA polymerases function

 addition of new nucleotides

DNA ligases function

stitching together (ligating) the newly created nucleotides to the existing strand

scaffolding proteins function in complex DNA repair

various roles → allow interaction and / or attraction of various repair proteins to sites of damage

EXCISION REPAIR PATHWAYS - mismatch repair (MMR) corrects: 

• single base mismatch 

• insertion / deletion

EXCISION REPAIR PATHWAYS - base excision repair (BER) corrects: 

• small base modifications

• abasic sites (AP sites)

• single strand (ss) breaks

EXCISION REPAIR PATHWAYS - nucleotide excision repair (NER) corrects: 

• bulky or helix distorting damage

• interstrand crosslinks

what does GGNER stand for?

global genome nucleotide excision repair

what is the damage recognition molecule in GGNER

XPC-RAD23B

(GGNERR = global genome nucleotide excision repair)

which molecules detect bloackage in Tx-coupled NER?

CSA/CSB

which polymerase is used in translation synthesis as form of DNA repair? What is unique about it?

Translesion polymerase

• only inserts 1 - 2 bases

• lacks proofreading ability → no endonuclease activity

• error prone → often inserts wrong base causing mutation

what is the trade-off taking place in Translation Synthesis as a form of DNA repair?

The cell is choosing a small risk (mutation) over a major risk (cell death from fork collapse)

• Benefit: avoids catastrophic DNA breaks and keeps replication going.

• Drawback: introduces base substitution mutations, increasing the chance of genetic errors.

consequences of unrepaired DSB

1. genomic instability

2. mutations

3. cell death

4. cancer.

Which sensor proteins recognise the ends of DSB and bind the DNA strand?

MRN and Ku70/Ku80 complexes

possible consequences of NHEJ

1. translocation of lost nucleotides on a damaged DNA strand

2. loss of nucleotides at the site of repair

3. interuption of gene expression

which DNA repair mechanism is primarily responsible for repairing DSB?

Homologous Recombination (HR)

• error free strategy

• can only occur S1/ S2 phase → requires sister chromatic

distinguish between NHEJ & HR

Feature	NHEJ	HR
Template required	❌ No	✅ Yes (sister chromatid)
Fidelity	❌ Error-prone (small deletions)	✅ High-fidelity
Cell cycle phase	All phases (G1, S, G2, M)	Only in S and G2 (requires a sister chromatid)
Speed	✅ Fast	❌ Slower
Common use	Quick fix, emergency repair	Clean, accurate repair of complex/ ‘unclean’ damage
Major risk	Translocations, mutations	Limited availability (needs template)

when is HR or NHEJ preferentially chosen?

HR = accuracy is vital and a template is available

NHEJ = speed is the priority.

What is Artemis role in NHEJ?

trims or modifies the DNA ends

which 2 molecules ligate the ends in NHEJ?

DNA ligase + XRCC4

role of RPA in DNA repair

used for Tx-coupled NER & HR

• coats the ssDNA

• Prevents the ssDNA from forming loops or being degraded

What is the DDR and what is its function??

• WHAT: a network of pathways the cell uses to detect, signal, and repair DNA damage.

• FUNCTION: It ensures genomic stability, and when damage is too severe, it can trigger cell cycle arrest, senescence, or apoptosis.

Key concept:
Damage → Sensor Proteins → Kinases → Cell Cycle Arrest → Repair OR Death

DDR - Single-Stranded DNA Damage (SSBs and replication fork stalling)

STRETCHES OF ssDNA SIGNAL DDR VIA ATR/CHK1 PATHWAY

1. DNA replication stalls due to damage (e.g. a DNA lesion).

2. Helicase continues, exposing single-stranded DNA (ssDNA).

3. RPA binds to exposed ssDNA → recruits ATRIP

4. ATRIP activates ATR kinase

5. ATR phosphorylates CHK1 → inactivates CDC25 phosphatases

6. CDC25 inactivation → CDK2 + Cyclin E can't function → intra-S phase arrest

SSB or replication fork stalling → RPA → ATRIP → ATR → CHK1 → CDC25 inactivated → S-phase arrest

DDR - Double-Stranded DNA Breaks (DSBs)

1. DSB is sensed by MRN complex and Ku70/80

2. Activates ATM kinase

3. ATM phosphorylates CHK2

4. CHK2 inactivates CDC25 phosphatases → blocks CDK activity → cell cycle arrest

DSB → MRN + Ku70/80 → ATM → CHK2 → CDC25 inactivated → cell cycle arrest

Role of p53 in DDR

• p53 is a tumour suppressor Tx factor protein that integrates upstream damage signals to the downstream pathways

• activated by phosphorylation of ATM / ATR / CHK1 and CHK2.

  • When phosphorylated → stabilises protein so p53 avoids degradation (not targeted by MDM2 = Ub ligase).

• stabilised p53 increases transcription of p21, which inhibits CDK2.

• This reinforces cell cycle arrest and gives time for repair or triggers apoptosis.

What are the hallmarks of cancer?

• early cancerous lesion – uncontrolled growth

• tumour progression = step-wise acquisition of various characteristics (hallmarks)

  • This is acquired by early genome instability

  • This instability attributed the cell adaptability to complex tumour environments

basic idea of DDR and Cancer

If DDR fails, DNA damage accumulates → genome instability → mutations in key genes (e.g. oncogenes and tumour suppressors) → cancer.

What is the multi-hit hypothesis in relation to Cancer?

Key idea = Cancer develops from the accumulation of multiple mutations over time, not just one.

EXPLAINS: Why cancer needs several mutations

• These mutations can affect tumour suppressors, oncogenes, DNA repair genes, etc.

• Each “hit” gives the cell a growth or survival advantage.

• Often visualised as stepwise progression (e.g. from normal → hyperplasia → dysplasia → carcinoma).

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What is the Mutator-phenotype hypothesis in relation to Cancer?

KEY IDEA: 1st mutation in DNA repair genes (e.g. MMR, NER) → increases overall mutation rate → step wise accumulation of mutations → higher risk of cancer (accelerates the multi-hit process)

EXPLAINS: How some cells get lots of mutations quickly

• Leads to genomic instability early in cancer development.

• As they accumulate these mutations they grow faster, survive longer and become 'fitter'

  • Attributes selective advantage that enables them to evade checkpoints

  • As they grow have ongoing adaptation = no blood supply, O2, nutrients

Examples of Inherited Disorders with Increased Cancer Risk (supports mutator hypothesis)

• Xeroderma pigmentosum → NER defect → XP gene defect → extreme UV sensitivity → reduced capacity to repair UV damage (pyrimidine dimers) → increased risk UV-induced skin cancers

• Lynch syndrome (HNPCC) → MMR defect → MutS/L gene prone mutation → ↓ DNA repair → ↑ mutation rate → ↑ risk of additional “hits” →colorectal & uterine cancers

what do the Multi-hit and Mutator Phenotype Hypothesis explain?

Multi-Hit Hypothesis = Why cancer needs several mutations → focus on any gene (oncogene, suppressor, etc.)

Mutator Phenotype = How some cells get lots of mutations quickly → focus on DNA repair genes

what causes genomic instability in sporadic (somatic) cancers? 

Oncogene activation → replicative stress → DSBs → DDR responds
But eventually: mutation in DDR → genomic chaos → cancer progression accelerates

• In sporadic cancers, the first mutations are often in proto-oncogenes → turned into oncogenes

• This leads to:

  • Increased cell division

  • Replication stress (too much or faulty DNA replication)

  • Resulting in DSBs and DNA damage

• more replication = more DNA damage = mutation in DDR genes = uncontrolled proliferation

• damage – stalled replication forks

• oncogenes – alterations to replication timing and progression

  • If have oncogenes that are just driving cell cycle forward without pausing or looking for damage = exposure of various fragments that are prone to being damaged = double strand breaks

Do oncogene induced tumour progression require the up or down regulation of the DDR?

Down-regulation of damage surveillance mechanisms (DDR).

oncogenes drive excessive proliferation → replicative stress

• normally DDR kicks in to kill cell, but cancer cell wants to keep growing

• therefore disable/ downregulate so cell can continue dividing

Key Difference: What activates ATM vs ATR?

ATM	ATR

Activated by double-strand breaks (DSBs)

Activated by single-stranded DNA (ssDNA) regions, especially at stalled replication forks

Sensor complex: MRN complex

Sensor: RPA-coated ssDNA recruits ATRIP

Major downstream target: CHK2

Major downstream target: CHK1

Cell Cycle Effect: Inhibits CDC25 → G1/S and G2/M arrest

Cell Cycle Effect: Inhibits CDC25 → Intra-S and G2/M arrest

main cell cycle effect of P53

stabilised by CHK1/2 → increases p21 expression → inhibits CDK → reinforces G1 and G2 checkpoints