DNA Structure, Replication & Repair

DNA is the molecular blueprint of life, and its structure, faithful duplication, and error-correction machinery form the bedrock of molecular biology — a perennial favourite in NEET PG. This chapter ties together Watson-Crick architecture, Chargaff's rules, the replication apparatus, and the three classical repair pathways whose failure underlies several named cancer syndromes.

Structure of DNA — the Watson-Crick model

DNA (deoxyribonucleic acid) is a polymer of deoxyribonucleotides, each composed of a nitrogenous base + deoxyribose sugar + phosphate. The bases are purines — adenine (A) and guanine (G) — and pyrimidines — cytosine (C) and thymine (T). RNA replaces thymine with uracil and uses ribose.

The Watson-Crick (1953) model describes B-DNA:

• Two antiparallel polynucleotide strands: one runs 5'→3', the complementary one 3'→5'.
• A right-handed double helix with the sugar-phosphate backbone outside and bases stacked inside.
• Strands held by complementary base pairing: A=T (2 hydrogen bonds), G≡C (3 hydrogen bonds). The extra H-bond makes GC-rich DNA more thermostable (higher melting temperature, Tm).
• Bases are connected to the backbone by N-glycosidic bonds; nucleotides within a strand are joined by 3'-5' phosphodiester bonds.
• Stabilised by hydrogen bonds and hydrophobic base-stacking interactions (the latter contribute most to overall stability).

High-yield: A pairs with T (2 H-bonds); G pairs with C (3 H-bonds). Higher G+C content → higher Tm → more heat-stable DNA.

Chargaff's rules

Erwin Chargaff observed, before the helix was solved, that:

1. A = T and G = C (molar equivalence of complementary bases).
2. Therefore purines = pyrimidines (A+G = T+C).
3. Base composition (A+T)/(G+C) ratio is species-specific and constant within a species but varies between species.

High-yield: Chargaff's rule (A=T, G=C) applies only to double-stranded DNA — not to single-stranded DNA or RNA.

B-DNA dimensions and forms

The classic Watson-Crick form is B-DNA. Memorise its parameters and compare with the alternative forms.

Feature	A-DNA	B-DNA (physiological)	Z-DNA
Handedness	Right	Right	Left
Helix diameter	~26 Å	~20 Å	~18 Å
Base pairs per turn	11	~10 (10.5)	12
Rise per bp	2.3 Å	3.4 Å	3.8 Å
Conditions	Dehydrated/RNA-DNA hybrids	Hydrated, dominant in vivo	GC-rich, alternating purine-pyrimidine; transcription regulation

High-yield: Z-DNA is the only left-handed form, has a zig-zag backbone, and occurs in GC-rich alternating sequences. B-DNA: 10 bp/turn, pitch 34 Å, rise 3.4 Å per base pair.

Higher-order packaging

In eukaryotes, ~2 metres of DNA is compacted into the nucleus by wrapping around histones. The fundamental unit is the nucleosome: ~146 bp of DNA wrapped 1.65 turns around a histone octamer (two each of H2A, H2B, H3, H4). H1 is the linker histone that binds linker DNA and stabilises higher-order folding. Histones are rich in lysine and arginine (positively charged), enabling tight binding to the negatively charged DNA backbone. Acetylation of histone lysines (by HATs) loosens chromatin → active transcription; deacetylation (HDACs) and DNA methylation silence genes.

DNA replication — overview

Replication is semiconservative (proved by Meselson-Stahl, 1958 using ¹⁵N density-gradient centrifugation): each daughter duplex retains one parental strand and one newly synthesised strand. It is bidirectional from each origin and proceeds in the 5'→3' direction only, using the parental strand as template. The substrate is dNTPs, and energy comes from cleavage of the pyrophosphate (PPi).

High-yield: All DNA polymerases synthesise DNA only 5'→3' and cannot initiate de novo — they need a free 3'-OH provided by an RNA primer made by primase.

Stepwise flow at the replication fork

Origin recognition → Helicase unwinds → SSB/RPA coats single strands → Topoisomerase relieves supercoiling → Primase lays RNA primer → Pol synthesises new strand → Primer removed & gap filled → Ligase seals nick.

1. Initiation at origin(s) of replication. Prokaryotes have a single origin (oriC); eukaryotes have multiple origins. Origin Recognition Complex (ORC) in eukaryotes.
2. Unwinding by helicase (DnaB in E. coli; MCM2-7 in eukaryotes), creating a replication fork.
3. Single-strand binding proteins (SSB in prokaryotes; RPA in eukaryotes) prevent reannealing and protect ssDNA.
4. Topoisomerases relieve torsional strain ahead of the fork. Topoisomerase II (DNA gyrase in bacteria) introduces negative supercoils; cuts both strands. Topoisomerase I cuts a single strand.
5. Primase (DnaG in prokaryotes) synthesises a short RNA primer providing the 3'-OH.
6. DNA polymerase extends the primer, adding dNTPs.
7. Primer removal, gap filling, and ligation.

Leading vs lagging strand & Okazaki fragments

Because polymerase works only 5'→3' but the two template strands are antiparallel:

• Leading strand is synthesised continuously toward the fork.
• Lagging strand is synthesised discontinuously, away from the fork, as short Okazaki fragments (~1000–2000 nt in prokaryotes; ~100–200 nt in eukaryotes), each needing its own RNA primer.

Feature	Leading strand	Lagging strand
Synthesis	Continuous	Discontinuous (Okazaki fragments)
Direction relative to fork	Toward fork	Away from fork
Number of primers	One	Many
Net direction	5'→3'	5'→3' (each fragment)

High-yield: Okazaki fragments are made on the lagging strand. Each fragment begins with an RNA primer that is later removed.

Prokaryotic vs eukaryotic replication machinery

Function	Prokaryote (E. coli)	Eukaryote
Main replicative polymerase	Pol III	Pol δ (lagging), Pol ε (leading)
Primer removal / repair	Pol I (5'→3' exonuclease, nick translation)	Pol δ + FEN1 + RNase H
Primase	DnaG	Pol α-primase
Helicase	DnaB	MCM2-7
SSB	SSB protein	RPA
Sliding clamp	β-clamp	PCNA
Topoisomerase	DNA gyrase (Topo II)	Topo I & II
Mitochondrial polymerase	—	Pol γ

High-yield: Pol I removes RNA primers (5'→3' exonuclease) and fills the gap — the basis of nick translation. Pol III is the chief synthetic enzyme. In eukaryotes, Pol γ replicates mitochondrial DNA.

Proofreading and fidelity

DNA polymerases (Pol III, Pol δ, Pol ε) possess 3'→5' exonuclease (proofreading) activity that removes mismatched nucleotides immediately after incorporation, reducing error rate dramatically. Pol I uniquely has both 3'→5' (proofreading) and 5'→3' exonuclease (primer removal) activities.

High-yield: 3'→5' exonuclease = proofreading; 5'→3' exonuclease (Pol I) = primer removal / nick translation. Don't confuse the two.

Telomeres and telomerase

Linear eukaryotic chromosomes face the end-replication problem — removal of the terminal lagging-strand primer leaves a 3' overhang that cannot be filled, shortening the chromosome each cycle. Telomerase, a reverse transcriptase (ribonucleoprotein) carrying its own RNA template, adds TTAGGG repeats to the 3' end. Telomerase is active in germ cells, stem cells, and ~85–90% of cancers (a hallmark of immortalisation) but largely inactive in somatic cells → progressive telomere shortening, replicative senescence, and the Hayflick limit.

High-yield: Telomerase is a reverse transcriptase; its reactivation is a hallmark of cancer. Telomere shortening underlies cellular ageing.

DNA repair mechanisms

Several pathways correct different lesion types. The three classic high-yield pathways are BER, NER, and MMR.

1. Base Excision Repair (BER)

• Corrects single damaged/abnormal bases (deamination, oxidation, alkylation, e.g. uracil from cytosine deamination, 8-oxoguanine).
• A specific DNA glycosylase recognises and removes the abnormal base → creates an AP (apurinic/apyrimidinic) site → AP endonuclease nicks the backbone → gap filled by Pol β and sealed by ligase.

2. Nucleotide Excision Repair (NER)

• Corrects bulky helix-distorting lesions: pyrimidine (thymine) dimers from UV light, and large chemical adducts.
• An oligonucleotide containing the lesion (~24–32 nt) is excised by endonucleases; Pol fills the gap; ligase seals.
• Defect → Xeroderma Pigmentosum (XP): extreme UV sensitivity, severe photosensitivity, freckling, and markedly increased skin cancers (BCC, SCC, melanoma) in sun-exposed areas.

3. Mismatch Repair (MMR)

• Corrects base-base mismatches and small insertion/deletion loops that escaped proofreading during replication.
• Key proteins: MSH2, MLH1 (and MSH6, PMS2). In bacteria, strand discrimination uses methylation (Dam methylase; the new strand is transiently unmethylated).
• Defect → Hereditary Non-Polyposis Colorectal Cancer (HNPCC / Lynch syndrome), marked by microsatellite instability (MSI).

Pathway	Lesion repaired	Key enzyme/protein	Defect disease
BER	Single abnormal base (deamination, oxidation)	DNA glycosylase, AP endonuclease	—
NER	Bulky lesions, UV thymine dimers	Excision nuclease (XP proteins)	Xeroderma pigmentosum
MMR	Base mismatches, indel loops	MSH2, MLH1	HNPCC (Lynch), MSI
HR / NHEJ	Double-strand breaks	BRCA1/2, RAD51 (HR); Ku, DNA-PK (NHEJ)	Breast/ovarian Ca, ataxia telangiectasia

High-yield mnemonic — "NUTS": NER repairs UV-induced Thymine dimers; defect causes Skin cancer (XP).

Double-strand break repair

• Homologous recombination (HR): error-free, uses sister chromatid as template; requires BRCA1/BRCA2, RAD51. BRCA mutations → hereditary breast and ovarian cancer; basis of PARP inhibitor (olaparib) synthetic lethality.
• Non-homologous end joining (NHEJ): error-prone, ligates broken ends directly; uses Ku70/80, DNA-PK, ligase IV.
• Ataxia telangiectasia results from ATM kinase mutation (defective DSB sensing) → cerebellar ataxia, oculocutaneous telangiectasia, radiosensitivity, lymphoma risk, raised alpha-fetoprotein.

High-yield: BRCA1/2 mutations impair homologous recombination → breast/ovarian cancer; PARP inhibitors exploit this defect (synthetic lethality).

Replication errors, mutations & cancer predisposition

Uncorrected replication errors and unrepaired damage become mutations (point mutations — transitions/transversions; frameshifts from insertions/deletions). Accumulation in proto-oncogenes and tumour-suppressor genes drives carcinogenesis. The DNA-repair-deficiency cancer syndromes are extremely high-yield:

Syndrome	Defective pathway/gene	Hallmark feature
Xeroderma pigmentosum	NER	UV sensitivity, skin cancers
HNPCC / Lynch	MMR (MSH2, MLH1)	Colorectal Ca, MSI
Hereditary breast/ovarian Ca	HR (BRCA1/2)	Breast, ovarian Ca
Ataxia telangiectasia	ATM (DSB sensing)	Ataxia, telangiectasia, lymphoma
Bloom syndrome	BLM helicase	Growth retardation, immunodeficiency, sister chromatid exchange
Fanconi anaemia	FANC genes (interstrand crosslink repair)	Pancytopenia, AML, congenital anomalies

High-yield: Pair each syndrome with its pathway — XP↔NER, Lynch↔MMR, BRCA↔HR. This single mapping yields multiple PYQ answers.

Inhibitors acting on DNA — pharmacology crossover

• Fluoroquinolones (ciprofloxacin) inhibit bacterial DNA gyrase (Topo II) and Topo IV.
• Etoposide inhibits eukaryotic Topo II; irinotecan/topotecan inhibit Topo I.
• Acyclovir / nucleoside analogues act as chain terminators (lack 3'-OH).
• Dideoxynucleotides (ddNTPs) terminate chain growth — the principle of Sanger sequencing.

High-yield: Chain-terminating ddNTPs (no 3'-OH) are the basis of Sanger dideoxy sequencing. Fluoroquinolones target bacterial gyrase.

Key differentials & conceptual contrasts

• DNA vs RNA: deoxyribose vs ribose; thymine vs uracil; double- vs single-stranded; stable vs labile.
• Transition vs transversion: transition = purine↔purine or pyrimidine↔pyrimidine; transversion = purine↔pyrimidine.
• Proofreading (3'→5' exo) vs MMR: proofreading acts during synthesis on the polymerase; MMR acts post-replication.
• Nucleosome vs solenoid: nucleosome (beads-on-a-string, 11 nm) → 30 nm solenoid fibre → higher-order loops.

Recently asked / exam angle

• "All DNA polymerases synthesise in which direction?" → 5'→3'.
• "Number of hydrogen bonds between G and C?" → 3.
• "Okazaki fragments are seen on which strand?" → Lagging.
• "Enzyme removing RNA primer in prokaryotes?" → DNA polymerase I (5'→3' exonuclease).
• "Which DNA is left-handed?" → Z-DNA.
• "Enzyme defective in xeroderma pigmentosum?" → NER (excision endonuclease).
• "Microsatellite instability is associated with defect in?" → Mismatch repair (Lynch/HNPCC).
• "Telomerase is which type of enzyme?" → Reverse transcriptase (RNA-dependent DNA polymerase).
• "Meselson-Stahl experiment proved?" → Semiconservative replication.
• "Base pairs per turn of B-DNA?" → ~10.
• "Enzyme replicating mitochondrial DNA?" → DNA polymerase γ.
• "Sliding clamp in eukaryotes?" → PCNA.

Rapid revision

1. DNA is antiparallel, right-handed (B-form), 10 bp/turn, rise 3.4 Å, pitch 34 Å.
2. A=T (2 H-bonds), G≡C (3 H-bonds); higher GC → higher Tm.
3. Chargaff: A=T, G=C, purines = pyrimidines — only in dsDNA.
4. Z-DNA is the only left-handed form (GC-rich, zig-zag backbone).
5. Replication is semiconservative (Meselson-Stahl), bidirectional, 5'→3' only.
6. Polymerases need an RNA primer (made by primase) — they cannot initiate de novo.
7. Leading strand continuous; lagging strand = Okazaki fragments.
8. Pol III synthesises; Pol I removes primer (5'→3' exo) and does nick translation; 3'→5' exo = proofreading.
9. Eukaryotic Pol δ/ε replicate nuclear DNA; Pol γ replicates mitochondrial DNA; PCNA is the sliding clamp.
10. Telomerase = reverse transcriptase adding TTAGGG; reactivated in cancers.
11. NER repairs UV thymine dimers → defect = xeroderma pigmentosum; MMR defect → Lynch/HNPCC + MSI; BER fixes single damaged bases.
12. BRCA1/2 (HR defect) → breast/ovarian cancer; PARP inhibitors exploit synthetic lethality; ATM defect → ataxia telangiectasia.