DNA Damage & Repair

What did Francis Crick later realize about DNA repair?

DNA is so precious that many distinct repair mechanisms must exist

What are the consequences of DNA damage?

• Cell death

• Cell cycle arrest

• Senescence

• Mutations (heritable nucleotide changes)

Why are mutations significant?

• Drive evolution and adaptation

• Can be deleterious, causing disease or cancer

How do mutations affect RAS genes?

• 20% of human tumors have activating RAS point mutations

• Mutations (codons 12, 13, 61) inhibit GTPase activity → RAS stays active (GTP-bound)

• Affects KRAS, NRAS, and HRAS isoforms

What are the endogenous sources of DNA damage?

• Replication errors

• Free radicals

• Spontaneous base deamination or depurination

What are the exogenous sources of DNA damage?

• Ionising radiation

• UV light

• Chemical carcinogens (e.g., benzo[a]pyrene, aflatoxin, anticancer drugs)

What are the two relationships between DNA damage and cancer?

• Causative: Damage leads to cancer (e.g., UV → skin cancer, smoking → lung cancer)

• Therapeutic: Chemotherapy and radiotherapy exploit DNA damage to kill cancer cells

What are common types of base modifications and their frequency per cell per day?

• Abasic (AP) sites: 2,000–10,000

• Cytosine → Uracil (deamination): 100–500

• Adenine → Hypoxanthine: 10–50

• 8-oxo-dG (oxidation): 100–500

• Alkylation damage (O6-methylguanine)

These cause mispairing and structural distortion of DNA

How does cigarette smoke damage DNA?

• Benzo[a]pyrene (BP) is converted by cytochrome P450 (CYP1A1) to BPDE

• BPDE binds guanine’s amine group → DNA adducts → mutations

How does alcohol cause DNA adducts?

• Ethanol → acetaldehyde (via ADH) → reacts with DNA forming:

  • N2-ethylidene-dG (weakly mutagenic)

  • 1,N2-propano-dG (highly mutagenic)

• High-risk groups: heavy drinkers with fast ADH or slow ALDH activity → oral & oesophageal cancer

What are the steps of Base Excision Repair?

1. DNA glycosylase removes damaged base → abasic (AP) site

2. AP endonuclease cleaves at AP site

3. DNA polymerase fills in missing nucleotide

4. DNA ligase seals the sugar-phosphate backbone

What lesions are repaired by NER?

Bulky distortions, e.g., UV-induced cyclobutane pyrimidine dimers (CPDs) and thymidine dimers

How frequent are UV-induced lesions?

About 40,000 damaged sites per hour in a skin cell under strong sunlight (UV 200–320 nm)

Describe the mechanism of NER

• Damage recognized by XPA + XPC complex

• DNA unwound by XPB/XPD helicases

• XPF/ERCC1 and XPG cut 5′ and 3′ sides of lesion

• DNA polymerase δ or ε fills gap

• DNA ligase seals the strand

What disease results from defective NER?

Xeroderma pigmentosum (XP)

• Caused by inherited XP gene mutations

• Leads to hypersensitivity to light and extreme UV-induced skin cancer susceptibility (melanoma, SCC)

What errors are corrected by Mismatch Repair (MMR)?

• Misincorporated nucleotides

• Small insertions or deletions (indels)

Describe the MMR mechanism

• MSH2/MSH6 detect mismatches

• Sliding clamp identifies a nick on the new strand

• Exonuclease removes mismatched section

• DNA polymerase δ/ε resynthesises DNA

• DNA ligase seals the strand

What happens when MMR fails?

• Microsatellite instability → accumulation of mutations

• HNPCC/Lynch syndrome (~3% of colon cancers) caused by MSH2/MLH1 mutations

• Cells may lose TGF-β receptor function, becoming resistant to growth inhibition

What causes Double-Strand Break (DSBs)?

• Ionising radiation (X-rays, γ-rays)

• Replication fork collapse

• Topoisomerase II inhibitors (e.g., doxorubicin/adriamycin)

Why are DSBs dangerous?

• Can cause cell death if unrepaired

• Cause mutations/chromosomal aberrations if misrepaired

Describe Non-Homologous End-Joining (NHEJ)

• Ku70/Ku80 bind DNA ends

• DNA ligase IV/XRCC4 joins the ends

• Error-prone but crucial (e.g., for V(D)J recombination in immunity)

Describe Homologous Recombination (HR)

• Accurate, template-directed repair.

• Steps:

  1. DNA end resection → ssDNA exposed.

  2. Rad51 forms nucleoprotein filament → homology search.

  3. Strand invasion & DNA synthesis.

  4. Resolution of Holliday junction.

• Key proteins: BRCA1, BRCA2

Germline DNA Repair Defects Table

Syndrome	Defective Mechanism	Instability	Cancer Predisposition
Xeroderma pigmentosum	NER	Point mutations	UV-induced skin cancer
Cockayne/Trichothiodystrophy	NER	Point mutations	None
Li-Fraumeni	DNA damage response	Chromosome aberrations	Various cancers
Ataxia-telangiectasia	DSB repair	Chromosome aberrations	Lymphomas
Nijmegen breakage	DSB repair	Chromosome aberrations	Lymphomas
BRCA1/2 mutations	HR	Chromosome aberrations	Breast/ovarian cancer
Werner	HR	Chromosome aberrations	Various cancers
Bloom	HR	Sister chromatid exchange	Leukemia/lymphoma
Fanconi anaemia	HR	Chromosome aberrations	AML, HNSCC
HNPCC/Lynch	MMR	Point mutations	Colorectal cancer

What did Pearl et al. (2015) show about repair pathway mutations?

Different cancer types have distinct frequencies of DNA damage response (DDR) mutations

How can DNA repair defects be exploited for therapy?

• Normal cells repair spontaneous replication breaks using HR and PARP1

• BRCA2-defective cells lack HR and depend on PARP1

• PARP1 inhibitors selectively kill BRCA2-negative cancer cells