Molecular Basis of Inheritance: DNA and Replication — Notes

Properties of a genetic material: it should (i) be able to generate its own replica; (ii) be stable chemically and structurally; (iii) provide scope for slow changes (mutation) required for evolution; (iv) be able to express itself as Mendelian characters. DNA meets these better than RNA: the 2′-OH of ribose makes RNA reactive and easily degraded, and thymine (instead of uracil) gives DNA extra stability. RNA can act as a catalyst and is thought to be the first genetic material (RNA world); DNA evolved from it.

Griffith (1928) — transformation. Streptococcus pneumoniae: S strain (smooth, with a polysaccharide capsule, virulent) and R strain (rough, no capsule, non-virulent). Live S → mouse dies; live R → lives; heat-killed S → lives; heat-killed S + live R → mouse dies, and live S bacteria are recovered. Some “transforming principle” from dead S cells had transformed R cells.

Avery, MacLeod and McCarty (1933–44) purified proteins, DNA and RNA from heat-killed S cells. DNA alone transformed R cells; proteases and RNases did not affect transformation, but DNase inhibited it. Conclusion: DNA is the hereditary material.

Hershey and Chase (1952) grew bacteriophages on media with radioactive phosphorus (³²P), which is in DNA but not protein, or radioactive sulphur (³⁵S), which is in protein but not DNA. Phages infected E. coli; blending removed the phage coats and centrifugation separated bacteria (pellet) from phage coats (supernatant). Bacteria infected with ³²P phages were radioactive; with ³⁵S phages the radioactivity stayed in the supernatant. So DNA, not protein, enters the bacteria: DNA is the genetic material.

Contributions: Friedrich Miescher (1869) isolated an acidic substance from the nuclei of pus cells, which he called nuclein (DNA). Erwin Chargaff: in double-stranded DNA the amount of adenine equals thymine and guanine equals cytosine (A = T, G = C; A + G = T + C); the ratio (A + T)/(G + C) varies between species. Maurice Wilkins and Rosalind Franklin produced X-ray diffraction data. James Watson and Francis Crick (1953) proposed the double helix model.

Polynucleotide chain. A nucleotide has a nitrogenous base, a pentose sugar and a phosphate. A nitrogenous base joined to the 1′ carbon of the sugar by an N-glycosidic linkage forms a nucleoside; a phosphate joined to the 5′-OH by a phosphoester linkage forms a nucleotide; nucleotides are joined by 3′–5′ phosphodiester linkages. Purines: adenine, guanine; pyrimidines: cytosine, thymine (DNA) and uracil (RNA).

Double helix: two polynucleotide chains with a sugar–phosphate backbone outside and bases inside; the chains are antiparallel (one 5′→3′, the other 3′→5′); A pairs with T by two hydrogen bonds, G with C by three; a purine always faces a pyrimidine, giving a uniform distance between the strands; the chains are coiled in a right-handed fashion; pitch 3.4 nm, about 10 bp per turn, 0.34 nm between base pairs; base pairs are stacked over one another, adding stability.

DNA vs RNA: DNA has deoxyribose, thymine and is usually double-stranded; RNA has ribose, uracil and is usually single-stranded.

Types of RNA: mRNA (provides the template for translation), tRNA (brings amino acids and reads the genetic code; adapter molecule) and rRNA (structural and catalytic role in ribosomes).

The distance between two consecutive base pairs is 0.34 nm, so the DNA of a typical mammalian cell (6.6 × 10⁹ bp) is about 2.2 m long — far longer than the nucleus (about 10⁻⁶ m). It must be packaged.

• Prokaryotes (e.g. E. coli) have no defined nucleus; their negatively charged DNA is held with some positively charged proteins in a region called the nucleoid, organised in large loops.
• Eukaryotes have a set of positively charged basic proteins, histones, rich in the basic amino acids lysine and arginine. Eight histone molecules form a histone octamer. Negatively charged DNA wraps around it to form a nucleosome, containing about 200 bp of DNA. Repeating nucleosomes form chromatin, which looks like beads on a string under the electron microscope; the chromatin fibre is coiled and condensed at metaphase to form chromosomes. Higher-level packaging requires additional proteins, the non-histone chromosomal (NHC) proteins.
• Euchromatin: loosely packed, stains light, transcriptionally active. Heterochromatin: densely packed, stains dark, inactive.

Francis Crick proposed the central dogma of molecular biology: genetic information flows from DNA → RNA → protein. DNA is replicated; its information is transcribed into RNA and translated into protein.

In some viruses the flow of information is in the reverse direction, from RNA to DNA. Retroviruses (such as HIV) carry the enzyme reverse transcriptase, which makes DNA from their RNA genome; the DNA can integrate into the host genome. This reverse transcription is an exception to the central dogma as first stated.

Watson and Crick proposed that the two strands separate and each acts as a template for a new complementary strand, so each daughter molecule has one parental (old) and one new strand: semi-conservative replication.

Meselson and Stahl (1958): E. coli grown for many generations in ¹⁵NH₄Cl (¹⁵N, the heavy isotope, as the only nitrogen source) were transferred to ¹⁴NH₄Cl. Samples taken at intervals were analysed by caesium chloride (CsCl) density-gradient centrifugation. After one generation (about 20 min) the DNA had a hybrid (intermediate) density; after two generations (40 min) there were equal amounts of hybrid and light DNA — as predicted by semi-conservative replication.

Taylor (1958): using radioactive thymidine with root tips of Vicia faba (broad bean) and autoradiography, Taylor showed that DNA in chromosomes also replicates semi-conservatively.

Machinery. Replication begins at an origin of replication. The two strands unwind at the replication fork. The main enzyme is DNA-dependent DNA polymerase, which uses a DNA template to polymerise deoxyribonucleotides; deoxyribonucleoside triphosphates serve as both substrates and energy source. It polymerises only in the 5′→3′ direction, so on one template the new strand is made continuously (leading strand) and on the other discontinuously in short pieces (Okazaki fragments, lagging strand), which are joined by DNA ligase. E. coli, with 4.6 × 10⁶ bp, completes replication in about 18 minutes: an average rate of about 2000 bp per second, with high accuracy. In eukaryotes, replication takes place in the S phase of the cell cycle.