DNA Structure, Replication & Repair

DNA is the molecule of heredity. Its structure, faithful replication, and accurate repair are fundamental to cell survival, genome stability, and the prevention of cancer. Errors in any of these processes underlie a wide range of genetic diseases and malignancies.

DNA Structure

DNA (deoxyribonucleic acid) is a double-stranded helix in which two antiparallel polynucleotide strands are held together by complementary base pairing. Adenine (A) pairs with thymine (T) via two hydrogen bonds; guanine (G) pairs with cytosine (C) via three hydrogen bonds. The antiparallel orientation means one strand runs 5'→3' while its complement runs 3'→5'. The double helix has a major groove and a minor groove — the major groove is wider and exposes more chemical information, making it the primary binding site for sequence-specific transcription factors.

In eukaryotes, DNA is packaged into chromatin. The nucleosome is the basic unit: ~147 bp of DNA wrapped ~1.65 turns around an octamer of histone proteins (two copies each of H2A, H2B, H3, H4). Histone H1 links adjacent nucleosomes. Post-translational modifications of histone tails regulate chromatin accessibility: acetylation (by HATs) generally opens chromatin (euchromatin, active transcription); deacetylation (by HDACs) compacts it (heterochromatin, gene silencing). Methylation of H3K4 marks active genes; H3K9 and H3K27 methylation marks repressed genes. DNA methylation at CpG dinucleotides is associated with gene silencing and is maintained through replication by DNMT1.

DNA Replication

Replication is semiconservative: each daughter molecule retains one parental strand and acquires one newly synthesised strand (demonstrated by Meselson and Stahl, 1958). In eukaryotes, replication initiates simultaneously at thousands of origins of replication (defined by ORC — origin recognition complex), allowing the entire ~3 billion bp genome to be copied in S phase (~8 hours). At each origin, the MCM helicase complex unwinds the double helix, creating bidirectional replication forks.

Key enzymes at the replication fork: Helicase (MCM2-7) unwinds the helix by breaking hydrogen bonds. Topoisomerase I and II relieve positive supercoiling ahead of the fork. Single-strand DNA binding proteins (RPA) stabilise exposed template strands. Primase synthesises short RNA primers (~10 nt) providing a 3'-OH for DNA polymerase. DNA polymerase δ (lagging strand) and ε (leading strand) synthesise DNA exclusively in the 5'→3' direction. The leading strand is synthesised continuously toward the fork. The lagging strand is synthesised discontinuously as Okazaki fragments (~100–200 nt in eukaryotes), each initiated by a new RNA primer. RNase H and FEN1 remove RNA primers; DNA polymerase δ fills the gaps; DNA ligase I seals nicks.

Telomeres — repetitive sequences (TTAGGG)n at chromosome ends — shorten with each division because the lagging strand cannot be fully replicated (end-replication problem). Telomerase, a ribonucleoprotein reverse transcriptase, uses its RNA template to extend telomeres, but is expressed only in germline cells, stem cells, and most cancer cells. Somatic cells progressively shorten telomeres, contributing to cellular senescence.

DNA Repair

The genome is under constant assault from endogenous (replication errors, reactive oxygen species, spontaneous depurination/deamination) and exogenous (UV radiation, ionising radiation, chemical carcinogens) sources of damage. Multiple repair pathways maintain genomic integrity:

Base excision repair (BER): corrects small base lesions (oxidised, deaminated, or methylated bases). A DNA glycosylase removes the damaged base; APE1 (AP endonuclease) nicks the backbone; polymerase β fills the gap; ligase seals.

Nucleotide excision repair (NER): removes bulky lesions such as UV-induced thymine dimers (cyclobutane pyrimidine dimers, CPDs) and 6-4 photoproducts. Defective NER causes xeroderma pigmentosum (XP) — extreme UV sensitivity and dramatically elevated skin cancer risk. NER involves ~30 proteins recognising the lesion, unwinding ~30 nt of DNA around the damage, excising the damaged oligonucleotide, and resynthesising using the opposite strand as template.

Mismatch repair (MMR): corrects replication errors (base mismatches, insertion-deletion loops). MutS homologues (MSH2, MSH6) recognise mismatches; MutL homologues (MLH1, PMS2) coordinate excision and resynthesis. Germline MMR mutations cause Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC) — elevated risk of colorectal, endometrial, ovarian, and other cancers.

Double-strand break (DSB) repair: DSBs are the most cytotoxic lesions. Two major pathways: (1) Homologous recombination (HR) — uses the sister chromatid as a template for error-free repair; requires BRCA1, BRCA2, and RAD51. Active in S and G2 phases. BRCA1/2 mutations cause hereditary breast and ovarian cancer syndrome. (2) Non-homologous end joining (NHEJ) — directly ligates broken ends via Ku70/Ku80, DNA-PKcs, and LIG4; error-prone (small insertions/deletions) but can operate throughout the cell cycle.

Clinical Correlations

PARP inhibitors (olaparib, niraparib) exploit synthetic lethality in BRCA-deficient tumours: cells lacking HR are dependent on PARP for single-strand break repair; PARP inhibition causes DSBs that cannot be repaired without BRCA → selective cancer cell death. Cisplatin forms intrastrand crosslinks; NER-deficient cancer cells cannot repair these, making them sensitive to platinum chemotherapy. Lynch syndrome screening: microsatellite instability (MSI) testing and immunohistochemistry for MMR proteins are now standard in colorectal cancer diagnosis, with implications for immunotherapy eligibility (high MSI tumours respond to pembrolizumab).