DNA Damage and Repair Mechanisms

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Quadrant I · e-Text Content

DNA Damage & Repair Mechanisms

A comprehensive study of how DNA sustains damage from endogenous and exogenous sources, and the remarkable molecular machinery that detects, signals, and corrects these lesions to maintain genomic integrity.

✓ Types of DNA damage ✓ Direct reversal ✓ BER & NER pathways ✓ MMR & DSB repair ✓ DNA damage checkpoints ✓ Diseases & Cancer

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1.1 Introduction: Why DNA Repair Matters

The DNA double helix is the most important molecule in biology — it carries the complete genetic instructions for every living organism. However, DNA is not chemically inert; it is constantly challenged by damaging agents both from within the cell and from the external environment.

It is estimated that each human cell sustains approximately 10,000–100,000 DNA lesions per day. Left unrepaired, these lesions can stall replication, cause mutations, or trigger cell death. To counteract this, cells have evolved a highly sophisticated DNA Damage Response (DDR) — a network of sensor proteins, signalling cascades, and repair enzymes that collectively maintain genomic integrity.

🔑 Central Dogma of DNA Repair DNA damage → Damage recognition → Signal transduction → Cell cycle arrest → Repair (or apoptosis if irreparable) → Cell cycle resumption. The entire DDR is orchestrated by kinases ATM and ATR.

⚡ Biological Significance • Unrepaired DNA damage → mutations (if replicated over) → cancer
• Persistent DSBs → chromosomal rearrangements → genomic instability
• Defective repair genes → inherited cancer syndromes (Xeroderma Pigmentosum, BRCA1/2, Lynch syndrome)
• Understanding DNA repair is the basis of cancer chemotherapy and radiotherapy

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1.2 Types of DNA Damage

DNA damage can be broadly classified into endogenous (arising from normal cellular processes) and exogenous (caused by environmental agents). The type of damage determines which repair pathway is deployed.

Endogenous Sources Spontaneous hydrolysis (depurination, deamination), reactive oxygen species (ROS) from mitochondrial respiration, replication errors (misincorporation, slippage), S-adenosylmethionine (SAM) mediated methylation

Exogenous Sources UV radiation (sunlight), ionising radiation (X-rays, γ-rays), chemical carcinogens (benzo[a]pyrene, aflatoxin), alkylating agents (nitrogen mustards), intercalating agents (ethidium bromide, acridine dyes), crosslinking agents (psoralen, mitomycin C)

Major categories of DNA lesions:

UV Radiation

Pyrimidine Dimers

UV-B (254–320 nm) crosslinks adjacent pyrimidines (T-T, T-C, C-C) on the same strand — Cyclobutane Pyrimidine Dimers (CPDs) and 6-4 photoproducts. Distort helix; block replication.

Alkylation

Alkylated Bases

Methyl or ethyl groups added to N7-G, O6-G, O4-T by alkylating agents (mustards, EMS, MNU). O6-methylguanine mispairs with T → G:C → A:T transitions. Highly mutagenic.

Oxidation

Oxidised Bases

ROS (OH•, O₂•⁻, H₂O₂) from metabolism oxidise bases. 8-oxoguanine (8-oxoG) is the most common — mispairs with A → G:C → T:A transversions. Also causes strand breaks.

Ionising Radiation

Double-Strand Breaks

X-rays and γ-rays directly break both phosphodiester backbones (DSBs) or generate ROS. DSBs are the most lethal DNA lesion. Even one unrepaired DSB can cause chromosomal rearrangements.

Spontaneous

Depurination & Deamination

Depurination: hydrolysis of glycosidic bond → AP (abasic) site (~10,000/cell/day). Deamination: C → U, 5-methylC → T, A → Hypoxanthine. Most common spontaneous lesion.

Intercalation

Insertions / Frameshifts

Flat aromatic molecules (EtBr, acridines, aflatoxin-G adducts) intercalate between base pairs → distort helix → cause insertion/deletion mutations during replication → frameshifts.

Single-Strand Breaks (SSBs) vs Double-Strand Breaks (DSBs) SSBs: one strand cut; other strand intact — used as template for repair; less dangerous. DSBs: both strands cut — no intact template; repaired by HR or NHEJ; most dangerous lesion.

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1.3 Direct Reversal of DNA Damage

The simplest form of DNA repair — the damage is chemically reversed by a single enzyme without excision of any nucleotides. No template is required and no gap is created.

① Photolyase (Photoreactivation) • Enzyme: DNA photolyase (light-dependent)
• Substrate: Cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts
• Mechanism: Photolyase binds the dimer in the dark; absorbs visible/near-UV light (300–500 nm) → excited FAD cofactor donates electron → dimer ring is split back into two separate pyrimidines
• Found in bacteria, yeast, plants; absent in placental mammals
• Humans use NER instead for UV damage

② O6-Methylguanine-DNA Methyltransferase (MGMT / AGT) • Enzyme: MGMT (suicide enzyme — alkyl-acceptor protein)
• Substrate: O6-methylguanine, O6-ethylguanine, O4-methylthymine
• Mechanism: MGMT irreversibly transfers the methyl group from O6 of guanine to a cysteine residue in its own active site → DNA restored, MGMT permanently inactivated (1 enzyme per repair event)
• Present in all organisms; important in cancer drug resistance (temozolomide)
• MGMT promoter hypermethylation → loss of MGMT → better response to alkylating chemotherapy in glioblastoma

③ AlkB Dioxygenases (Oxidative Demethylation) • Enzyme: AlkB (E. coli); ALKBH2, ALKBH3 (humans)
• Substrate: 1-methyladenine (1-meA), 3-methylcytosine (3-meC)
• Mechanism: AlkB uses α-ketoglutarate and O₂ as cosubstrates → oxidative removal of methyl group via hydroxymethyl intermediate → formaldehyde released → base restored
• Important for repair of RNA damage as well as DNA

Key Features of Direct Reversal Most efficient repair — no strand discontinuity, no new nucleotides required, no risk of introducing errors. However, limited to specific lesions (mainly alkylation and UV dimers).

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1.4 Base Excision Repair (BER)

BER is the primary pathway for repairing small, non-helix-distorting base lesions such as oxidised, deaminated, or alkylated bases, and AP sites. It operates through two sub-pathways: Short-Patch BER (1 nt replaced) and Long-Patch BER (2–10 nt replaced).

Key Enzymes 1. DNA glycosylase — recognises and excises damaged base (monofunctional or bifunctional)
2. AP endonuclease (APE1) — cleaves phosphodiester bond 5' to AP site → 5' nick
3. DNA polymerase β (Pol β) — Short-patch: fills 1-nt gap; also has lyase activity (removes 5' dRP)
4. DNA polymerase δ/ε + PCNA + RFC — Long-patch: strand displacement synthesis (2–10 nt)
5. FEN1 — flap endonuclease; removes displaced 5' flap in long-patch BER
6. DNA ligase III/XRCC1 — seals nick (short-patch); DNA ligase I (long-patch)

Step-by-step BER mechanism:

Original damaged DNA: 5'—A—T—U—G—C—T—A—3' ← Uracil from C deamination 3'—T—A—A—C—G—A—T—5' Step 1: UNG (Uracil DNA Glycosylase) removes uracil: 5'—A—T—[AP]—G—C—T—A—3' ← Abasic (AP) site created 3'—T—A— A —C—G—A—T—5' Step 2: APE1 cuts 5' to AP site: 5'—A—T—|—G—C—T—A—3' ← Nick (single-strand break) 3'—T—A— A—C—G—A—T—5' Step 3: Pol β fills gap using complementary strand as template: 5'—A—T—C—G—C—T—A—3' ← Correct C inserted 3'—T—A— A—C—G—A—T—5' Step 4: DNA Ligase III/XRCC1 seals nick: 5'—A—T—C—G—C—T—A—3' ← Repaired! ✓ 3'—T—A— A—C—G—A—T—5'

Examples of DNA Glycosylases and Their Substrates • UNG (Uracil DNA Glycosylase) — removes uracil (from deamination of C)
• OGG1 (8-oxoG DNA glycosylase) — removes 8-oxoguanine (oxidation)
• AAG/MPG — removes 3-methyladenine, hypoxanthine (alkylation)
• MBD4 — removes thymine from G:T mispairs (deamination of 5-methylC)
• NEIL1/NEIL2 — removes ring-opened purines, oxidised pyrimidines

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1.5 Nucleotide Excision Repair (NER)

NER repairs bulky, helix-distorting lesions — primarily UV-induced pyrimidine dimers, as well as chemical adducts (benzo[a]pyrene-G, cisplatin intrastrand crosslinks). It excises a 25–30 nt oligonucleotide containing the damage. NER operates via two sub-pathways differing in damage recognition.

Two Sub-Pathways 1. Global Genome NER (GG-NER) — scans the entire genome; XPC-RAD23B detects helix distortion
2. Transcription-Coupled NER (TC-NER) — activated when RNA Pol II stalls at a lesion; CSA, CSB (Cockayne syndrome proteins) recruited; faster, prioritises transcribed strands

Core NER mechanism (shared steps after recognition):

Step-by-Step NER 1. Damage recognition: XPC-RAD23B (GG-NER) or stalled RNA Pol II + CSB (TC-NER) recognise lesion
2. Verification: TFIIH complex (XPB + XPD helicases) opens DNA ~25 bp around the lesion
3. Dual incision: XPG cuts 3' side (+3 to +8); XPF-ERCC1 cuts 5' side (−15 to −24) → releases ~25–30 nt oligonucleotide containing the damage
4. RPA, PCNA: stabilise single-stranded gap; recruit repair polymerases
5. Gap filling: DNA Pol δ, Pol ε, or Pol κ fills the gap using the intact complementary strand
6. Ligation: DNA Ligase I or III seals the nick → repair complete

Feature	GG-NER	TC-NER
Recognition	XPC-RAD23B (whole genome)	Stalled RNA Pol II + CSB/CSA
Speed	Slower	Faster (transcribed strands)
Template bias	None	Transcribed strand preferred
Disease if defective	Xeroderma Pigmentosum (XP)	Cockayne Syndrome (CS)

Xeroderma Pigmentosum (XP) Autosomal recessive; defective NER (mutations in XPA–XPG or XPV). Extreme UV sensitivity, 10,000× increased skin cancer risk, neurological degeneration. Seven complementation groups (XP-A to XP-G) + variant (XP-V; defective Pol η). A classic example of a DNA repair disease.

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1.6 Mismatch Repair (MMR)

MMR corrects base-base mismatches and small insertion/deletion loops (IDLs) that escape the proofreading activity of replicative DNA polymerases. It is tightly coupled to replication and increases replication fidelity by ~100-fold (on top of polymerase proofreading).

Key MMR Proteins (Human) • MutSα (MSH2-MSH6) — recognises single base mismatches and small IDLs
• MutSβ (MSH2-MSH3) — recognises larger IDLs (2–4 nt)
• MutLα (MLH1-PMS2) — endonuclease; recruited by MutS; nicks the newly-synthesised strand
• EXO1 — excises the error-containing strand from nick to mismatch
• PCNA, RFC, RPA — processivity factors and ssDNA stabilisers
• DNA Pol δ — re-synthesises the excised region
• DNA Ligase I — seals the nick

How Does MMR Know Which Strand to Excise? In bacteria: GATC methylation (by Dam methylase) marks the old (template) strand → newly synthesised strand is transiently unmethylated → MutH nicks the unmethylated strand → repair of the new (incorrect) strand. In eukaryotes: strand discrimination achieved through nicks left by the replication machinery (Okazaki fragments on lagging strand; nick at 3' end of new strand on leading strand). PCNA sliding clamp helps direct repair.

Lynch Syndrome (HNPCC) — MMR Defect Most common hereditary colorectal cancer syndrome. Caused by germline mutations in MLH1, MSH2, MSH6, or PMS2. Hallmark: microsatellite instability (MSI) — accumulation of mutations in repetitive sequences due to failed MMR. Also predisposes to endometrial, ovarian, stomach cancers. Tumours with MSI have many neoantigens → better immunotherapy response.

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1.7 Double-Strand Break (DSB) Repair

DSBs are the most genotoxic form of DNA damage. Even a single unrepaired DSB can trigger cell death or chromosomal rearrangements. Cells repair DSBs by two major pathways: Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ).

① Homologous Recombination (HR) Uses the sister chromatid (or homologous chromosome) as a template → accurate, error-free. Predominant in S and G2 phases when a sister chromatid is available.

Key steps:
1. DNA end processing (resection): MRN complex (MRE11-RAD50-NBS1) + CtIP resect 5' ends → 3' single-stranded overhangs
2. RPA coating: RPA binds and stabilises ssDNA overhangs
3. RAD51 filament: BRCA2 loads RAD51 onto ssDNA → nucleoprotein filament forms
4. Strand invasion: RAD51 filament searches for homologous sequence on sister chromatid → D-loop formation
5. DNA synthesis: 3' end extended using sister chromatid as template
6. Resolution: Holliday junction formed and resolved → accurate repair

② Non-Homologous End Joining (NHEJ) Directly rejoins broken DNA ends — does not require a homologous template. Active in all cell cycle phases (dominant in G1). Fast but potentially mutagenic (small insertions/deletions at junction).

Key steps:
1. End recognition: Ku70-Ku80 heterodimer binds and protects DNA ends
2. DNA-PKcs recruitment: DNA-PKcs (serine/threonine kinase) activated → phosphorylates H2AX, itself, other targets
3. End processing: Artemis (endonuclease) trims overhangs; Pol μ/λ fill gaps
4. Ligation: XRCC4-DNA Ligase IV-XLF complex ligates the two ends
5. Result: DSB rejoined — may have small indels at the junction

Feature	HR	NHEJ
Template required	Yes (sister chromatid)	No
Cell cycle phase	S and G2	All phases (dominant G1)
Accuracy	Error-free	May cause small indels
Key proteins	MRN, RPA, RAD51, BRCA1/2	Ku70/80, DNA-PKcs, XRCC4, Lig IV
Speed	Slower	Faster
Disease if defective	BRCA1/2 → breast/ovarian cancer	DNA-PKcs → SCID, radiosensitivity

BRCA1 and BRCA2 in HR BRCA1 promotes end resection (5'→3'); BRCA2 directly loads RAD51 onto RPA-coated ssDNA — essential for strand invasion. Germline mutations in BRCA1/2 → impaired HR → reliance on error-prone NHEJ → genomic instability → 60–80% lifetime breast cancer risk, high ovarian cancer risk. Basis of PARP inhibitor therapy (synthetic lethality).

Alt-NHEJ / Microhomology-Mediated End Joining (MMEJ) A backup DSB repair pathway; uses 5–25 nt microhomologies flanking the break. Highly mutagenic — generates deletions. Associated with chromosomal translocations in cancer. Requires PARP1, MRE11, Pol θ (POLQ).

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1.8 DNA Damage Checkpoints

Checkpoints are surveillance mechanisms that halt cell cycle progression when DNA damage is detected, providing time for repair before replication or mitosis. They are the central mediators of the DNA Damage Response (DDR).

Master Kinases: ATM and ATR • ATM (Ataxia Telangiectasia Mutated) — activated by DSBs; phosphorylates H2AX (γ-H2AX), CHK2, BRCA1, p53, MDM2
• ATR (ATM and Rad3-related) — activated by RPA-coated ssDNA (at stalled replication forks or after resection); phosphorylates CHK1, RPA, FANCI/D2
Both are PIKK-family kinases that coordinate repair with cell cycle arrest.

G1/S Checkpoint (Most Important) DSB detected → ATM activated → phosphorylates CHK2 → CHK2 phosphorylates CDC25A (promotes its degradation) and p53 → p53 stabilised (MDM2 phosphorylated by ATM → MDM2 degraded) → p53 activates p21 transcription → p21 inhibits CDK2-Cyclin E → CDK2 cannot phosphorylate Rb → E2F blocked → S-phase entry HALTED. Also: CHK2 phosphorylates CDC25A → CDK4/6 inhibited → G1 arrest maintained.

Intra-S Phase Checkpoint Slows replication during S phase if damage detected. ATM/CHK2 and ATR/CHK1 phosphorylate CDC25A → degraded → CDK2 inactive → replication origins cannot fire → replication forks slow/stall. Prevents replication through damaged template.

G2/M Checkpoint Prevents mitosis with damaged DNA. ATM → CHK2; ATR → CHK1 → both phosphorylate CDC25C → CDC25C degraded/exported → CDK1-Cyclin B (MPF) remains inactive → mitosis blocked. WEE1 kinase also phosphorylates and inhibits CDK1.

p53 — Guardian of the Genome p53 (TP53) is the most mutated gene in human cancer (~50% of all cancers). Normally kept at low levels by MDM2-mediated ubiquitination and proteasomal degradation. Upon DNA damage:
ATM/ATR → phosphorylate p53 at Ser15 and Ser20 → blocks MDM2 binding → p53 stabilised → activates: p21 (cell cycle arrest), PUMA/NOXA (apoptosis), DDB2 (NER), GADD45 (DNA repair co-factor). Decides cell fate: repair or apoptosis.

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1.9 Translesion Synthesis (TLS)

When a replication fork stalls at a DNA lesion, Translesion Synthesis (TLS) allows specialised DNA polymerases to bypass the lesion — a tolerance mechanism rather than true repair. TLS polymerases have larger, more accommodating active sites than replicative polymerases, enabling synthesis past damaged bases at the cost of lower fidelity.

Key TLS Polymerases (Y-family) • Pol η (Eta) — bypasses CPDs accurately (T-T dimers → inserts A-A); defective in XP variant (XP-V)
• Pol ι (Iota) — bypasses abasic sites and 8-oxoG; low fidelity
• Pol κ (Kappa) — bypasses benzo[a]pyrene-dG adducts; extends mispairs
• Rev1 — inserts C opposite abasic sites; scaffolds other TLS polymerases
• Pol ζ (Zeta, B-family) — Rev3-Rev7; extends mispaired termini; error-prone extension

Mechanism of Polymerase Switching 1. Replicative Pol δ/ε stalls at lesion → uncoupling of helicase and polymerase
2. PCNA ubiquitinated at Lys164 by RAD6-RAD18 (mono-Ub) → recruits TLS polymerase
3. TLS Pol inserts nucleotide(s) across the lesion (error-prone)
4. Pol ζ extends the TLS product
5. Replicative polymerase resumes synthesis downstream
Poly-ubiquitination (by Ubc13-Mms2-SHPRH/HLTF) triggers template switching (error-free)

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1.10 DNA Repair Diseases and Cancer Connection

Defects in DNA repair genes are directly responsible for several human genetic syndromes characterised by cancer predisposition, neurodegeneration, developmental defects, and immune deficiency.

Disease	Defective Pathway	Gene(s)	Clinical Features
Xeroderma Pigmentosum (XP)	NER	XPA–XPG, XPV (Pol η)	UV sensitivity, skin cancer (10,000× risk), neurodegeneration
Cockayne Syndrome (CS)	TC-NER	CSA (ERCC8), CSB (ERCC6)	Photosensitivity, premature ageing, neurological defects, dwarfism; NO cancer
Trichothiodystrophy (TTD)	NER/TFIIH	XPB, XPD, TTDA	Brittle hair (sulphur-deficient), photosensitivity, intellectual disability
Lynch Syndrome (HNPCC)	MMR	MLH1, MSH2, MSH6, PMS2	Hereditary colorectal, endometrial, ovarian cancers; microsatellite instability
Hereditary Breast/Ovarian Cancer	HR (DSB repair)	BRCA1, BRCA2	60–80% breast cancer risk, 40% ovarian cancer risk; PARP inhibitor sensitive
Ataxia Telangiectasia (AT)	DSB signalling	ATM	Cerebellar ataxia, telangiectasia, lymphoma risk, radiosensitivity, immunodeficiency
Nijmegen Breakage Syndrome	DSB repair (MRN)	NBS1 (Nibrin)	Microcephaly, immunodeficiency, lymphoma, radiosensitivity
Fanconi Anaemia (FA)	ICL repair	FANCA–FANCW (22 genes)	Bone marrow failure, congenital abnormalities, AML, squamous cell carcinoma
Werner Syndrome	HR, BER	WRN (RecQ helicase)	Premature ageing (progeroid), sarcoma, type 2 diabetes

DNA Repair and Cancer Therapy • PARP inhibitors (olaparib, rucaparib) — synthetic lethality in BRCA1/2 mutant tumours; block BER backup → DSBs → cell death in HR-deficient cells
• Temozolomide (alkylating agent) — effective in MGMT-silenced glioblastoma
• Cisplatin/oxaliplatin — crosslinking agents; effective in NER-deficient tumours
• MSI-High tumours (MMR-deficient) — respond to PD-1/PD-L1 checkpoint immunotherapy (pembrolizumab approved)

Quadrant II · e-Tutorial / Interactive Simulations

Visualise the Repair Pathways

Interactive step-by-step pathway walkthroughs, a DNA damage simulator, and a historical timeline of DNA repair discoveries.

🎛️ DNA damage simulator 🔬 Pathway explorer 📅 Discovery timeline

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DNA Damage & Repair Simulator — Click a scenario

Normal nucleotides

Damaged bases

Repaired bases

Newly synthesised

Gap / missing

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Repair Pathway Step-by-Step Explorer

Base Excision Repair (BER) — Target: Small non-bulky lesions (8-oxoG, Uracil, AP sites)

Damage Recognition

A lesion-specific DNA glycosylase (e.g., OGG1 for 8-oxoG; UNG for uracil) scans DNA by diffusion along the minor groove, flips the damaged base into its catalytic pocket, and cleaves the N-glycosidic bond → base removed, AP site created.

Enzyme: DNA Glycosylase

AP Site Incision

APE1 (AP Endonuclease 1) recognises the AP site and cleaves the phosphodiester bond 5' to the abasic site → 3'-OH and 5'-dRP (deoxyribose phosphate) ends generated → single-strand nick.

Enzyme: APE1

Gap Filling (Short Patch: 1 nt)

DNA Pol β inserts the correct nucleotide into the 1-nt gap (using the opposite strand as template). Pol β's lyase domain simultaneously removes the 5'-dRP group. This is the predominant sub-pathway in post-mitotic cells.

Enzyme: DNA Pol β

Nick Sealing

XRCC1 scaffolds the complex; DNA Ligase IIIα seals the remaining nick → covalently continuous, repaired DNA strand restored.

Enzyme: Ligase III/XRCC1

Nucleotide Excision Repair (NER) — Target: Bulky helix-distorting lesions (CPDs, cisplatin adducts)

Lesion Recognition

GG-NER: XPC-RAD23B detects helix distortion. TC-NER: RNA Pol II stalls at lesion; CSB binds; UVSSA-USP7 stabilises complex; XPA and RPA verify damage.

XPC-RAD23B / CSB/CSA

Helix Opening (TFIIH)

TFIIH (10-subunit complex; contains XPB and XPD helicases) unwinds ~25 bp around the lesion. XPA verifies chemical damage; RPA stabilises the bubble and positions other factors.

TFIIH (XPB + XPD)

Dual Incision

XPG cleaves 3' side (+3 to +8 from lesion). XPF-ERCC1 cleaves 5' side (−15 to −24). A 25–30 nt oligonucleotide containing the lesion is released. XPG must be present (but need not cut) before XPF-ERCC1 can cut 5'.

XPG (3') + XPF-ERCC1 (5')

Gap Filling & Ligation

PCNA + RFC clamp loader. DNA Pol δ or Pol ε (or Pol κ in TC-NER) fills the 25–30 nt gap. DNA Ligase I (or III/XRCC1) seals the nick → perfectly restored DNA.

Pol δ/ε → Ligase I

Mismatch Repair (MMR) — Target: Replication errors (mismatches, IDLs)

Mismatch Recognition

MutSα (MSH2-MSH6) slides along DNA and recognises base-base mismatches and 1-nt IDLs. MutSβ (MSH2-MSH3) recognises larger IDLs (2–4 nt). MutS homologs bend DNA upon binding, inducing a conformational change.

MutSα / MutSβ

MutL Recruitment & Strand Discrimination

MutSα/β recruits MutLα (MLH1-PMS2). PCNA and RFC at the replication nick direct MutLα to nick the newly synthesised (incorrect) strand. MutLα's endonuclease activity (PMS2 subunit) nicks the error strand.

MutLα (MLH1-PMS2)

Excision of Error-Containing Strand

EXO1 (5'→3' exonuclease) degrades the nicked strand from the strand discrimination nick past the mismatch → single-stranded gap. RPA stabilises the gap. Excision can also occur in the 3'→5' direction (EXO1-independent).

EXO1 → gap formation

Re-synthesis & Ligation

DNA Pol δ (with PCNA/RFC) fills the gap using the template (correct) strand. DNA Ligase I seals the nick → error-free DNA with correct base at the previously mismatched position.

Pol δ → Ligase I

Homologous Recombination (HR) — Target: DSBs in S/G2 phase

DSB Recognition & MRN Binding

MRN complex (MRE11-RAD50-NBS1) rapidly localises to DSBs. MRE11 has endo and exonuclease activity; RAD50 has ATPase and coiled-coil domains that tether broken ends; NBS1 recruits ATM.

MRN + ATM activation

End Resection

MRN + CtIP initiate short-range resection (endonucleolytic clipping). EXO1 or BLM-DNA2 extend resection to generate long 3' ssDNA overhangs. BRCA1 promotes resection. RPA coats the ssDNA to prevent hairpin formation.

CtIP/EXO1/BLM-DNA2

RAD51 Nucleofilament Formation

BRCA2 (with PALB2 and BRCA1) displaces RPA and loads RAD51 onto the 3' ssDNA → RAD51 nucleoprotein filament (ATP-bound). BRCA2 makes 8 BRC repeats that each bind one RAD51 monomer.

BRCA2 loads RAD51

Strand Invasion & D-loop

RAD51 filament searches the genome for sequence homology (sister chromatid). Upon finding a match, it invades the duplex → D-loop (displacement loop). The 3'-OH of the invading strand serves as a primer for DNA synthesis.

RAD51 strand invasion

DNA Synthesis & Resolution

DNA Pol δ/η extends from D-loop. SDSA (most common): newly synthesised strand dissociates, anneals to resected end, gap filled, ligated → non-crossover outcome. DSBR pathway → Holliday junction → resolution (crossover or non-crossover).

Pol δ/η → Ligation

Non-Homologous End Joining (NHEJ) — Target: DSBs in all cell cycle phases

End Recognition by Ku

Ku70-Ku80 heterodimer (ring-shaped) rapidly threads onto and binds double-stranded DNA ends with very high affinity. Ku protects DNA ends from excessive nucleolytic degradation and recruits DNA-PKcs.

Ku70-Ku80 heterodimer

DNA-PKcs Activation

DNA-PKcs (catalytic subunit; 460 kDa PIKK kinase) binds Ku at DNA ends → activated → autophosphorylates (T2609, S2056 clusters) → phosphorylates H2AX (γ-H2AX), RPA, and other repair factors. DNA-PKcs on opposing ends interact to synapse the two broken ends.

DNA-PKcs activation

End Processing

Artemis (endonuclease activated by DNA-PKcs) trims 5' overhangs and opens hairpins. Pol μ and Pol λ (X-family) perform limited gap filling or add nucleotides to 3' overhangs. Polynucleotide kinase/phosphatase (PNKP) ensures 5'-phosphate and 3'-OH ends.

Artemis / Pol μ / Pol λ

Ligation

XRCC4-DNA Ligase IV complex (stabilised by XLF/Cernunnos and PAXX) directly ligates the processed ends. XLF bridges XRCC4 molecules on opposing ends. Result: DSB rejoined — may have small insertions or deletions at the junction.

XRCC4-Lig IV-XLF

Direct Reversal — Target: Specific alkylation / UV damage (no excision needed)

Photolyase — UV Dimer Reversal

DNA photolyase binds CPDs in the dark (no light required for binding). Upon absorbing visible/near-UV light (300–500 nm), excited flavin cofactor (FADH•) transfers an electron to the cyclobutane ring → ring opens → two normal thymines restored. One enzymatic event per dimer; no strand break.

Photolyase + visible light

MGMT — Alkyl Group Transfer

MGMT (O6-methylguanine-DNA methyltransferase) recognises O6-meG in the major groove. It directly transfers the methyl group from O6-G to a specific cysteine residue (Cys145) in its own active site. MGMT is permanently inactivated — a "suicide enzyme." One MGMT molecule per repair event.

MGMT (suicide enzyme)

AlkB — Oxidative Demethylation

AlkB dioxygenase (humans: ALKBH2/3) uses Fe(II), O₂, and α-ketoglutarate as cosubstrates to oxidise the N-methyl group → unstable hydroxymethyl intermediate → spontaneously releases formaldehyde → base fully restored. Not a suicide enzyme — regenerated catalytically.

AlkB / ALKBH2/3

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Historical Timeline of DNA Repair Discoveries

1949 — Photoreactivation discovered

Albert Kelner and Renato Dulbecco independently discovered that UV-damaged bacteria could be repaired by visible light — the first DNA repair mechanism described.

1963 — Uvr genes discovered in E. coli

Setlow and Carrier demonstrated that UV-irradiated bacteria excise thymine dimers — laying the groundwork for Nucleotide Excision Repair discovery.

1968 — NER mechanism elucidated

Philip Hanawalt, Paul Howard-Flanders, and colleagues described the excision-repair pathway in E. coli — incision, excision, re-synthesis, ligation.

1974 — Xeroderma Pigmentosum linked to NER defect

Cleaver and colleagues showed XP cells are defective in DNA repair after UV irradiation — first inherited human disease linked to DNA repair deficiency.

1983 — Mismatch Repair mechanism (MutHLS)

Methyl-directed mismatch repair pathway characterised by Modrich and colleagues in E. coli — later extended to eukaryotic MMR and linked to Lynch syndrome.

1994 — BRCA1 and BRCA2 cloned

Hall et al. (1994) and Wooster et al. (1995) cloned BRCA1 and BRCA2 respectively — hereditary breast/ovarian cancer genes later shown to function in HR.

1994–2000 — Lynch syndrome linked to MMR

Fishel, Kolodner, Bhatt, Vogelstein et al. identified MLH1, MSH2, and other MMR genes as the cause of HNPCC/Lynch syndrome and microsatellite instability.

2015 — Nobel Prize in Chemistry

Tomas Lindahl, Paul Modrich, and Aziz Sancar awarded the Nobel Prize for their mechanistic studies of BER, MMR, and NER respectively — the highest recognition for DNA repair science.

2018 — PARP inhibitor approved for BRCA cancers

Olaparib (AstraZeneca) first approved by FDA for BRCA-mutant breast and ovarian cancer — clinical proof of synthetic lethality concept and translation of DNA repair biology to therapy.

Quadrant III · Self-Assessment

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MCQs with instant feedback, drag-and-drop pathway matching, and fill-in-the-blank exercises aligned to B.Sc. Zoology university exam patterns.

📝 15 MCQs with explanations 🧩 Drag & Drop ✏️ Fill in the Blanks

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Match: Repair Pathway to its Key Feature

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Drag each label to the correct description:

BER

NER

MMR

NHEJ

TLS

Direct Reversal

MGMT

Repairs 8-oxoG and uracil via DNA glycosylase:

Excises 25–30 nt oligo containing pyrimidine dimer:

Corrects replication mismatches; defective in Lynch syndrome:

Error-free DSB repair using sister chromatid template:

Rapid DSB repair; Ku70-Ku80 binds ends; may cause indels:

Pol η bypasses CPDs; PCNA ubiquitination required:

No excision; restores damaged DNA in single chemical step:

Suicide enzyme; transfers methyl from O6-methylguanine:

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Fill in the Blanks

Complete the sentences

Quadrant IV · Resources & References

Life Cycle of Antheraea mylitta