DNA damage and Repair

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Molecular Biology · B.Sc. Zoology · Unit V

DNA Damage
& Repair Mechanisms

A comprehensive interactive module on how living cells detect, respond to, and correct damage to their genetic material.

Base Excision Repair Nucleotide Excision Repair Mismatch Repair Double-Strand Break Repair SOS Response Photoreactivation

BN

Dr Bhabesh Nath

Assistant Professor, Dept. of Zoology
B N College Autonomous, Dhubri

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Introduction to DNA Damage & Repair

Understanding why cells must constantly monitor and repair their DNA — and the consequences when repair fails.

🧬

What is DNA Damage?

Any alteration in the chemical structure or nucleotide sequence of DNA that deviates from the normal double-helical form.

⚠️

Why does it matter?

Unrepaired DNA damage leads to mutations, genomic instability, cancer, aging, and cell death.

🔄

Rate of Damage

Human cells suffer ~10,000–100,000 DNA lesions per day from endogenous sources alone.

🛠️

Repair Systems

Cells have evolved at least 6 major repair pathways, each specialized for specific types of lesions.

Sources of DNA Damage

🌿 Endogenous (Internal) Sources ▾

• Hydrolysis — spontaneous loss of bases (depurination ~5,000/cell/day) or deamination of cytosine to uracil (~100–500/cell/day)
• Oxidative damage — reactive oxygen species (ROS) from cellular metabolism attack guanine (→ 8-oxoguanine) and other bases
• Replication errors — DNA polymerase misincorporates ~1 wrong nucleotide per 10⁵ bases; mismatch repair corrects most
• Topoisomerase failures — abortive topoisomerase reactions can leave strand breaks
• Alkylating agents — S-adenosylmethionine (SAM) can transfer methyl groups to DNA bases

☀️ Exogenous (External) Sources ▾

• UV radiation (UV-B, 260–280 nm) — creates cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts between adjacent pyrimidines
• Ionising radiation (X-rays, γ-rays) — directly breaks phosphodiester bonds; generates ROS causing oxidised bases and strand breaks
• Chemical mutagens — aflatoxin B1 (fungal toxin), benzo[a]pyrene (cigarette smoke), nitrogen mustards form bulky adducts
• Alkylating agents — MNNG, EMS add alkyl groups to N and O atoms of bases
• Intercalating agents — ethidium bromide, acridine dyes insert between base pairs causing frameshift mutations

🔬 Consequences of Unrepaired Damage ▾

• Point mutations — substitution of one base pair with another; may alter protein function
• Frameshift mutations — insertion or deletion shifts the reading frame, altering all downstream codons
• Chromosomal aberrations — deletions, inversions, translocations resulting from double-strand breaks
• Apoptosis — extensive damage triggers programmed cell death as a protective mechanism
• Carcinogenesis — mutations in proto-oncogenes and tumour suppressor genes (e.g., TP53, BRCA1/2) drive cancer
• Aging — accumulation of somatic mutations and epigenetic changes over time contribute to ageing phenotypes

⚠️ Xeroderma Pigmentosum, Cockayne Syndrome, and Fanconi Anaemia are human genetic diseases caused by defects in DNA repair genes, dramatically illustrating the importance of these pathways.

🔑 Key Concepts to Remember

• DNA damage is inevitable — cells deal with thousands of lesions per day
• Damage ≠ Mutation: damage is a physical/chemical alteration; mutation is a heritable change in sequence
• Repair pathways evolved in parallel with DNA itself; virtually all organisms possess them
• The choice of repair pathway depends on the lesion type and cell cycle stage
• Cell cycle checkpoints are activated by damage sensors to allow time for repair before replication or division

Types of DNA Damage

Explore the major categories of DNA lesions. Click each tab to learn about mechanism, examples, and biological significance.

🔓

Depurination (Apurinic Sites)

Cause: Spontaneous hydrolysis of glycosidic bond

Depurination is the most common form of spontaneous DNA damage. The N-glycosidic bond linking a purine base (adenine or guanine) to the deoxyribose sugar undergoes hydrolysis, releasing the base and creating an apurinic (AP) site.

• Occurs ~5,000 times per mammalian cell per day
• AP sites are non-instructional — DNA polymerase stalls or inserts adenine opposite (A-rule), causing transversion mutations
• Also termed an abasic site — the sugar-phosphate backbone remains intact
• If unrepaired, leads to deletion mutations during next replication
• Pyrimidines (cytosine, thymine) are much less susceptible — depyrimidination is ~5× less frequent

📌 Repair: Base Excision Repair (BER) via AP endonuclease and DNA polymerase β.

Frequency

Very High

🔁

Deamination of Bases

Cause: Spontaneous or chemical removal of amino group

Deamination converts amino groups (–NH₂) on bases into carbonyl groups (=O), chemically transforming one base into another:

• Cytosine → Uracil: Most common (~100–500/cell/day); if unrepaired, causes C·G → T·A transitions
• 5-methylcytosine → Thymine: Particularly mutagenic because thymine is a normal base, harder to detect; C·G → T·A hotspots (CpG sites)
• Adenine → Hypoxanthine: Hypoxanthine pairs with cytosine → A·T → G·C transitions
• Guanine → Xanthine: Xanthine still pairs with cytosine; less mutagenic but can block replication
• Nitrous acid (HNO₂) and bisulfite are classic chemical agents that cause deamination

📌 Uracil-DNA glycosylase (UNG) initiates BER to remove uracil from DNA.

Frequency

Moderate

☀️

UV-Induced Photoproducts

Cause: UV-B radiation (260–280 nm wavelength)

UV radiation causes the most mutagenic exogenous DNA damage by forming covalent bonds between adjacent pyrimidine bases on the same DNA strand:

• Cyclobutane Pyrimidine Dimers (CPDs): Comprise ~75% of UV lesions; four-membered ring between C5-C6 bonds of adjacent thymines (T^T); distort helix and block replication
• 6-4 Photoproducts (6-4PPs): Covalent bond between C6 of 5′ base and C4 of 3′ base; ~25% of UV lesions; more mutagenic and distorting than CPDs
• Both types predominantly occur at TC and CC sequences (pyrimidine dimers)
• Cause "C → T" or "CC → TT" signature mutations in skin cancers (UV signature)
• Block RNA polymerase transcription as well as DNA replication

⚠️ Defective NER leading to accumulation of UV lesions causes Xeroderma Pigmentosum — patients have ~1000-fold higher risk of skin cancer.

Mutagenicity

High

⚡

Oxidative DNA Damage

Cause: Reactive oxygen species (ROS) from metabolism, radiation

ROS including hydroxyl radical (•OH), superoxide (O₂•⁻), and hydrogen peroxide (H₂O₂) attack all four DNA bases and the sugar-phosphate backbone:

• 8-oxoguanine (8-oxo-dG): Most studied oxidative lesion; mispairs with adenine → G·C to T·A transversions; ~10,000 formed/cell/day
• Thymine glycol: Replication-blocking lesion from thymine oxidation
• Formamidopyrimidine (FAPY): Ring-opened purine; blocks replication and is mutagenic
• Strand breaks: •OH attacks the sugar, causing single-strand breaks
• Mitochondrial DNA is more susceptible to oxidative damage (closer to ROS-producing electron transport chain, less histone protection)

📌 The MutM/OGG1 glycosylase removes 8-oxoguanine; MutY/MUTYH removes adenine misincorporated opposite 8-oxoG.

Frequency

High

🧪

Alkylation Damage

Cause: Alkylating agents — endogenous SAM, exogenous mustards, MNNG

Alkylating agents transfer alkyl groups (methyl, ethyl, etc.) to electronegative atoms of DNA bases and the phosphate backbone:

• O⁶-methylguanine (O6-MeG): Most mutagenic — pairs with thymine instead of cytosine → G·C to A·T transitions; repaired by the direct-reversal enzyme MGMT
• N7-methylguanine: Most abundant alkylation product (~80%); relatively stable but can lead to depurination
• N3-methyladenine: Blocks replication (cytotoxic); repaired by AlkA/AAG glycosylases
• Nitrogen mustards (bifunctional): Cross-link two guanines either intrastrand or interstrand, blocking both replication and transcription
• Cisplatin (cancer drug) forms similar intrastrand cross-links, exploiting the cancer cell's inability to repair them

📌 Ada protein in E. coli is a DNA methyltransferase that directly removes methyl groups — it also activates the adaptive response to alkylation.

Mutagenicity

High

✂️

DNA Strand Breaks

Cause: Ionising radiation, ROS, abortive topoisomerase, replication stress

Phosphodiester bonds in the DNA backbone can be broken, producing single-strand breaks (SSBs) or the more dangerous double-strand breaks (DSBs):

• Single-Strand Breaks (SSBs): ~10,000–1,000,000/cell/day; generally repaired rapidly using the intact complementary strand as template
• Double-Strand Breaks (DSBs): ~10–50/cell/day from endogenous sources; both strands cut within ~10–20 bp; most dangerous type — loss of template information
• One Gray of ionising radiation produces ~20–40 DSBs per mammalian cell
• DSBs can cause chromosomal translocations, deletions, and fusions if misrepaired
• DSBs are also programmed events: meiotic recombination (SPO11 enzyme), V(D)J recombination in immune cells, and class-switch recombination

⚠️ A single unrepaired DSB can kill a cell or, if misrepaired, lead to chromosomal rearrangements driving cancer.

Severity

Extreme

🔀

Replication Mismatches & Insertions/Deletions

Cause: DNA polymerase errors, strand slippage at repetitive sequences

Despite proofreading, DNA polymerases occasionally incorporate wrong nucleotides or slip on repetitive templates:

• Base-base mismatches: G·T, A·C, G·G etc. — arise from tautomeric shifts in bases or oxidised bases (e.g., 8-oxoG·A)
• Insertion-deletion loops (IDLs): DNA polymerase slippage on repetitive sequences (microsatellites); creates extra bases or gaps in newly synthesised strand
• Microsatellite instability (MSI) is a hallmark of mismatch repair deficiency; seen in ~15% of colorectal, endometrial cancers
• Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC) caused by germline mutations in MMR genes MLH1, MSH2, MSH6, PMS2
• The overall uncorrected error rate of DNA replication is ~1 mistake per 10⁹–10¹⁰ base pairs, thanks to MMR

📌 MMR corrects ~99% of mismatches remaining after polymerase proofreading.

Frequency

Moderate

DNA Repair Mechanisms

Six major repair pathways — each specialised for different types of lesions. Click any card to expand detailed steps.

1

Base Excision Repair

BER — Small base lesions

Repairs single chemically modified bases (oxidised, deaminated, alkylated). Works through lesion-specific glycosylases that flip the damaged base out of the helix.

1. DNA glycosylase recognises and excises damaged base
2. AP endonuclease nicks the backbone at AP site
3. DNA polymerase fills the gap (short-patch or long-patch)
4. DNA ligase seals the nick

UNG OGG1 APE1 Pol β XRCC1 Ligase III

2

Nucleotide Excision Repair

NER — Bulky helix-distorting lesions

Removes bulky DNA adducts (UV dimers, chemical adducts) by cutting out a ~25–30 nucleotide single-stranded patch containing the lesion.

1. XPC-RAD23B recognises helix distortion (GG-NER)
2. TFIIH helicase unwinds DNA around lesion
3. XPG and XPF-ERCC1 make dual incisions
4. Pol δ/ε fills gap; ligase seals

XPC TFIIH/XPD XPA XPG XPF-ERCC1 RPA

3

Mismatch Repair

MMR — Replication errors

Corrects base-base mismatches and insertion/deletion loops arising from DNA polymerase errors. Must distinguish the new (error-containing) from the parental strand.

1. MutS (MSH2-MSH6) recognises mismatch
2. MutL (MLH1-PMS2) recruits excision machinery
3. MutH (bacteria) or strand discrimination signals (eukaryotes) identify new strand
4. Exonuclease excises error-containing stretch; Pol δ resynthesises

MSH2 MSH6 MLH1 PMS2 EXO1 PCNA

4

Homologous Recombination

HR — DSB repair (accurate)

High-fidelity repair of DSBs using a homologous template (sister chromatid). Operates in S/G2 phase when a sister chromatid is available.

1. MRN complex detects DSB; CtIP resects to generate 3′ ssDNA
2. RPA coats ssDNA; BRCA2 loads RAD51
3. RAD51 filament invades homologous duplex (strand invasion)
4. D-loop extension, branch migration, Holliday junction resolution

MRN BRCA1/2 RAD51 RPA RAD54 Gen1/Mus81

5

Non-Homologous End Joining

NHEJ — DSB repair (fast)

Rapid but error-prone repair of DSBs by direct ligation of broken ends. Dominant pathway in G1 phase and post-mitotic cells. Can cause small insertions/deletions.

1. Ku70/Ku80 heterodimer binds and protects broken ends
2. DNA-PKcs kinase is recruited and activated
3. Artemis nuclease trims incompatible ends
4. XRCC4-Ligase IV complex ligates the ends

Ku70/80 DNA-PKcs Artemis XRCC4 Lig IV XLF

6

Photoreactivation & Direct Reversal

Direct enzymatic repair

Simplest repair — damage reversed directly without excision. Photolyase uses visible light energy; MGMT uses its own cysteine residue as acceptor.

1. Photolyase binds CPD or 6-4PP lesion in dark
2. Visible light (300–500 nm) excites chromophore (MTHF)
3. Electron transfer breaks the cyclobutane ring
4. Normal bases restored; photolyase released (humans lack CPD photolyase)

Photolyase MGMT AlkB

SOS Response (Prokaryotes)

🆘 What is the SOS Response? ▾

The SOS response in E. coli is an inducible global response to extensive DNA damage that upregulates ~40 genes involved in repair, replication restart, and tolerance.

• Inducing signal: Single-stranded DNA (ssDNA) coated with RecA protein — formed at stalled replication forks or processed DSBs
• LexA repressor: Normally represses SOS genes by binding to SOS boxes (operator sequences) in their promoters
• RecA filament stimulates LexA autocleavage → derepression of ~40 SOS genes
• SOS polymerases (Pol IV/DinB, Pol V/UmuD'C): Error-prone translesion synthesis polymerases induced; can bypass lesions that block Pol III, but at cost of mutagenesis
• sulA gene: Encodes cell division inhibitor (blocks FtsZ ring) — halts division to allow repair time
• Recovery: As damage is repaired, ssDNA decreases → RecA filaments disassemble → LexA re-accumulates → SOS genes repressed again

⚠️ The SOS response is a last resort — it saves the cell but at the cost of increased mutagenesis. "Better to be alive and mutated than dead and pristine."

🔄 Translesion Synthesis (TLS) ▾

TLS is a DNA damage tolerance mechanism — it does not remove the lesion but allows replication to continue past it using specialised low-fidelity polymerases:

• The stalled replicative polymerase (Pol III/Pol δ) is replaced by a Y-family TLS polymerase via PCNA ubiquitination
• Pol η (eta): Accurately bypasses CPD dimers (T^T → AA); mutated in Xeroderma Pigmentosum variant (XP-V)
• Pol ι (iota): Bypasses abasic sites and 8-oxoG; low fidelity on undamaged templates
• Pol κ (kappa): Bypasses benzo[a]pyrene-guanine adducts
• Rev1: Inserts C opposite abasic sites and G; acts as scaffold for other TLS polymerases
• After bypass, the replicative polymerase resumes normal synthesis (polymerase switching)

Key Enzymes in DNA Repair

A structured reference of major repair enzymes, their functions, organisms, and associated pathways.

Enzyme / Protein	Organism	Pathway	Function	Substrate
UNG / UDG	All organisms	BER	Uracil-DNA glycosylase; excises uracil	Uracil in DNA
OGG1 / MutM	Eukaryotes / Bacteria	BER	Bifunctional glycosylase/lyase; removes 8-oxoguanine	8-oxoG·C
AAG / AlkA	Human / E. coli	BER	N-methylpurine glycosylase; removes alkylated bases	3-MeA, hypoxanthine
APE1 / Xth	Human / E. coli	BER	AP endonuclease; incises 5′ of AP site	Abasic (AP) sites
DNA Pol β	Eukaryotes	BER	Gap-filling polymerase; removes 5′ dRP	1-nt gap at AP site
XRCC1-Lig III	Eukaryotes	BER / SSB	Scaffold/nick ligation	Nicked DNA
XPC-RAD23B	Eukaryotes	GG-NER	Damage recognition; helix distortion sensor	UV dimers, bulky adducts
CSA / CSB	Eukaryotes	TC-NER	Coupling of stalled RNA Pol II to NER	Transcription-blocking lesions
TFIIH (XPD/XPB)	Eukaryotes	NER	ATP-dependent helicase; unwinds DNA ~30 bp	Lesion-containing duplex
XPA	Eukaryotes	NER	Damage verification; scaffold; binds RPA	Damaged DNA + RPA
XPG	Eukaryotes	NER	3′ endonuclease; cuts 6 nt 3′ of lesion	Unwound lesion
XPF-ERCC1	Eukaryotes	NER	5′ endonuclease; cuts 20–24 nt 5′ of lesion	Unwound lesion
MutS / MSH2-MSH6	All organisms	MMR	Mismatch recognition; ATPase sliding clamp	Base-base mismatches, IDLs
MutL / MLH1-PMS2	All organisms	MMR	Endonuclease; coordinates repair	MutS-mismatch complex
MutH	E. coli only	MMR	Nicks hemimethylated GATC on new strand	Hemimethylated GATC
MRN (MRE11-RAD50-NBS1)	Eukaryotes	HR / DSB sensing	DSB sensor; nuclease; tethers broken ends; activates ATM	DSBs
RAD51	Eukaryotes	HR	Forms nucleoprotein filament; catalyses strand invasion	ssDNA/homologous duplex
BRCA2	Eukaryotes	HR	Mediator; loads RAD51 onto RPA-coated ssDNA	RAD51, ssDNA
Ku70/Ku80	Eukaryotes	NHEJ	DSB end-binding; recruits DNA-PKcs	DSB ends
DNA-PKcs	Vertebrates	NHEJ	Serine/threonine kinase; phosphorylates repair factors	Ku-bound DSB ends
XRCC4-Lig IV	Eukaryotes	NHEJ	Ligation complex; seals blunt/compatible ends	DSB ends
Photolyase (CPD)	Bacteria, plants, some eukaryotes	Photoreactivation	Uses light energy to split CPDs	Cyclobutane pyrimidine dimers
MGMT / Ada (O6-MT)	All organisms	Direct reversal	Methyl acceptor; removes O6-Me from guanine (suicide enzyme)	O6-methylguanine
AlkB (ALKBH)	Bacteria / Eukaryotes	Direct reversal (oxidative)	Oxidative demethylase; removes N1-MeA, N3-MeC	N-alkylated bases
RecA / RAD51	Bacteria / Eukaryotes	HR / SOS	Homologous strand pairing; LexA co-protease activity (RecA)	ssDNA, dsDNA
Pol η (eta)	Eukaryotes	TLS	Accurate bypass of CPDs; Y-family polymerase	T^T dimers (template)

🔑 Enzyme Pattern Summary

• BER enzymes are highly damage-specific (one glycosylase per lesion type) but share the downstream AP endonuclease-to-ligase cascade
• NER is versatile — recognises helix distortion rather than specific chemistry, so repairs many different bulky lesions
• MMR is strand-specific — must distinguish template from newly synthesised strand (GATC methylation in bacteria; strand-discontinuities in eukaryotes)
• HR requires a sister chromatid template → restricted to S/G2; NHEJ is template-independent → works throughout the cell cycle
• MGMT and photolyase are "suicide enzymes" in that they can only perform one reaction — MGMT permanently accepts the methyl group and is degraded

DNA Damage & Repair Pathway Map

An overview of how different lesion types connect to the appropriate repair pathways — the cell's decision-making logic.

📌 Pathway choice depends on: (1) Type of lesion, (2) Phase of cell cycle, (3) Availability of sister chromatid template, (4) Extent of damage (mild → repair; severe → apoptosis).

Self-Test: DNA Damage & Repair

Test your understanding with these questions. Click an option to see if you are correct, along with an explanation.

0

Score

0

Answered

12

Total

Glossary of Key Terms

Search and browse important terminology used in DNA damage and repair biology.

DNA Damage & Repair — Interactive e-Content Module
Authored by Dr Bhabesh Nath, Assistant Professor, Department of Zoology,
B N College Autonomous, Dhubri | Published at zoollogys.co.in
For educational use — B.Sc. Zoology (Molecular Biology)