Describe the structure of DNA. Add a note on its denaturation and melting temperature.

Structure of DNA

• A polymer of deoxyribonucleotides, each consisting of a nitrogenous base, deoxyribose and a phosphate group, joined by 3′–5′ phosphodiester bonds
• Bases — the purines adenine and guanine (double-ringed) and the pyrimidines cytosine and thymine (single-ringed)
• WATSON and CRICK double helix — two antiparallel strands (one running 5′ to 3′, the other 3′ to 5′) wound into a right-handed helix about a common axis
• The sugar-phosphate backbone lies outside, hydrophilic and negatively charged, with the bases stacked inside
• Chargaff rules — A = T and G = C, so purines equal pyrimidines; the ratio (A+T)/(G+C) varies between species
• Base pairing by hydrogen bonds — A pairs with T by two bonds; G pairs with C by three
• Dimensions of B-DNA — one complete turn every 3.4 nm containing 10 base pairs, so adjacent bases are 0.34 nm apart; diameter 2 nm. major and minor grooves allow proteins to read the sequence without unwinding it
• Forms — B (the physiological right-handed form), A (right-handed, dehydrated) and Z (left-handed, with a zigzag backbone)

Denaturation and Melting Temperature

Heating, extremes of pH, urea or formamide break the hydrogen bonds between the strands → The phosphodiester backbone is not broken — denaturation is a separation, not a degradation → The two strands separate — denaturation or melting → The stacked bases are exposed, and absorbance at 260 nm increases by up to 40% — the hyperchromic effect → Tm (melting temperature) is the temperature at which half the DNA has become single-stranded → On slow cooling the complementary strands re-form — renaturation or annealing, with a corresponding hypochromic effect

• Tm rises with GC content, because of the third hydrogen bond, and also with increasing salt concentration and length of the molecule
• Tm falls with extremes of pH, urea, formamide and low ionic strength
• Hybridisation — single strands from different sources anneal if their sequences are complementary, which is the principle underlying Southern and Northern blotting, DNA probes, microarrays and PCR

Higher Order Structure

• DNA is wound around histone octamers (two each of H2A, H2B, H3 and H4) with about 146 base pairs per nucleosome, giving the "beads on a string" appearance; H1 is the linker histone
• Nucleosomes coil into a 30 nm fibre, then into loops and finally the condensed chromosome — a packing ratio of about 10,000-fold, which is what allows two metres of DNA to fit in a nucleus
• Histones are strongly basic (rich in lysine and arginine), so they bind the acidic phosphate backbone electrostatically
• Euchromatin is loosely packed and transcriptionally active; heterochromatin is condensed and inactive
• Supercoiling is managed by topoisomerases, which are the targets of quinolone antibiotics and of etoposide and irinotecan

Applied Aspects

• PCR depends directly on denaturation and annealing — heating to separate the strands, cooling to let primers anneal, and extension by a thermostable polymerase; the annealing temperature is chosen from the primer Tm
• Absorbance at 260 nm measures nucleic acid concentration, and the 260/280 ratio assesses protein contamination — about 1.8 for pure DNA and 2.0 for RNA
• Quinolones and several anticancer drugs act on topoisomerases, exploiting the fact that DNA must be transiently cut and resealed to be replicated
• Histone modification and DNA methylation regulate gene expression without altering the sequence — the basis of epigenetics, and the target of several newer anticancer drugs
• Ultraviolet light causes thymine dimers, repaired by nucleotide excision repair; its failure causes xeroderma pigmentosum, with extreme photosensitivity and early skin cancer