DNA replication

The three models of replication

Before the mechanism was known, three models were proposed. The conservative model said the original double helix stayed intact and an entirely new one was built alongside it. The dispersive model said the new molecules were patchworks, with stretches of old and new sequence scattered along both strands. The semi-conservative model, which Watson and Crick put forward in 1953 alongside their double helix, said the two strands separate, each acts as a template, and each new molecule contains one original strand and one new one.

Describe the drawing

A family tree of DNA molecules over two rounds of replication. The parent molecule at the top is two original strands. Arrows lead to two granddaughter rows: each of the two molecules in generation one is one original strand paired with one new strand, and of the four molecules in generation two, two still contain an original strand while two are made entirely of new strands.

The term semi-conservative refers to each daughter molecule: half of each is conserved from the parent. State it that way rather than as 'half the DNA is conserved', which is imprecise and, after several generations, incorrect.

Semi-conservative replication

Replication in which the two strands of the parent molecule separate and each acts as a template, so every daughter molecule contains one original strand and one newly synthesised strand.

Template strand

A strand of DNA whose base sequence determines the sequence of the new strand built against it.

Strand separation and synthesis

In a eukaryotic cell, DNA is replicated during S phase of interphase, before the cell divides. Replication begins when DNA helicase travels along the molecule and breaks the hydrogen bonds between paired bases. The two strands separate and the helix unwinds, exposing the bases. Name the bonds broken: hydrogen bonds, between the paired bases. The phosphodiester bonds along each backbone are covalent and are not broken, which is why the strands separate intact.

Free DNA nucleotides, already present in the nucleoplasm, then align against the exposed bases by complementary base pairing. An adenine on the template attracts a thymine nucleotide and nothing else; a cytosine attracts a guanine. The specificity of the pairing is what makes the copy accurate; it follows from the chemistry of the bases.

DNA polymerase then catalyses the condensation reactions that join the aligned nucleotides into a continuous strand, forming a phosphodiester bond between each one and the next. The free nucleotides arrive as nucleoside triphosphates, so the energy for each bond comes from the two extra phosphates being released as the bond forms.

Describe the drawing

A replication fork drawn as a sideways Y. The still-paired double helix is on the right, with helicase marked at the junction and an arrow showing the fork moving into it. Along the upper separated template a single long arrow points toward the fork, labelled the leading strand, with its five prime end behind it and its three prime end at the tip. Along the lower template three short arrows point away from the fork, the Okazaki fragments of the lagging strand.

DNA polymerase can only build a strand in the 5′ to 3′ direction, because it can only attach a new nucleotide to a free 3′ hydroxyl group. The two template strands are antiparallel, so the enzyme cannot travel the same way along both. On one template it moves in the direction the fork is opening, and produces one long unbroken leading strand. On the other it must work away from the fork, so it waits for a stretch of template to be exposed, copies backwards along it, then jumps forward and does it again. The result is the lagging strand: a series of short pieces called Okazaki fragments, typically 100 to 200 nucleotides long in eukaryotes, which DNA ligase later joins into one continuous strand.

The commonest confusion here is thinking the lagging strand is built 3′ to 5′. It is not. Both new strands are built 5′ to 3′ without exception. What differs is the direction of travel relative to the fork, and that is what forces one of them into fragments.

One footnote on an A-level simplification. DNA polymerase cannot start a strand from nothing, only extend one, so in the full account every new strand, the leading strand included, begins with a short RNA primer laid down by an enzyme called primase. On the lagging strand each fragment starts with its own primer, and the primers are removed and replaced with DNA before ligase seals the joins. Most courses leave the primers out; if yours names primase, this is where it fits.

The Meselson-Stahl experiment

In 1958 Matthew Meselson and Franklin Stahl found a way to tell the three models apart by weight. They grew E. coli for many generations in a medium whose only nitrogen source was the heavy isotope ¹⁵N. Nitrogen is in every base, so the bacteria built DNA that was measurably denser than normal. They then transferred the culture to a medium containing only ordinary ¹⁴N, so every nucleotide made from that point on was light, and sampled the DNA after each round of division.

Spinning the extracted DNA in a caesium chloride solution at very high speed sets up a density gradient, and each DNA molecule settles at the depth matching its own density. Heavy DNA sinks furthest. Light DNA stays highest. A hybrid molecule, one heavy strand and one light, settles halfway between.

Describe the drawing

Three columns of centrifuge tubes, one column for each proposed model of replication, with three tubes down each column for the starting DNA and the first and second generations. The conservative column keeps a heavy band throughout. The semi-conservative column shows a single intermediate band after one generation and two bands, intermediate and light, after two. The dispersive column shows a single band that drifts upward and never splits.

After one generation there was a single band, and it sat exactly halfway between the heavy and light positions. That result eliminates the conservative model, which predicts that the original heavy molecule survives intact and an entirely light molecule is built beside it, giving two bands, one heavy and one light, and no intermediate band. A conservative mechanism cannot produce a molecule of mixed density, because it never places old and new material in the same molecule.

That result alone does not distinguish semi-conservative from dispersive, because a patchwork molecule of half-old, half-new sequence would also be of intermediate density. The second generation separates them. The semi-conservative model predicts two bands: half the molecules still contain one heavy strand and stay intermediate, while half are now entirely light. The dispersive model predicts one band again, this time three quarters of the way toward light, because every molecule is still an even mixture. Two distinct bands appeared, which eliminates the dispersive model.

Replication errors and mutation

Human DNA replication leaves roughly one uncorrected error per billion base pairs copied, after DNA polymerase's own proofreading and the cell's mismatch repair have corrected most errors. Across 3.1 billion base pairs that still leaves several new errors in every cell division.

An uncorrected error is a mutation: a change in the base sequence of DNA. A base substitution swaps one base for another. An insertion or deletion adds or removes bases; within a coding region, adding or removing a number not divisible by three shifts the reading of every triplet downstream, which is usually far more damaging, while a whole number of triplets leaves the frame intact. Mutations also arise from mutagens such as ultraviolet light, some chemicals in tobacco smoke and ionising radiation, not only from errors in replication.

Mutation

A change in the base sequence of DNA.

Substitution

A mutation in which one base is replaced by another.

Deletion

A mutation in which a base is lost; within a coding region, losing a number of bases not divisible by three shifts the reading of every triplet that follows.

Most mutations have no effect. Some occur in non-coding stretches of DNA. Some change a triplet into another triplet coding for the same amino acid, a consequence of the degeneracy of the genetic code. Some change an amino acid in a region of the protein where the substitution does not alter the folded shape. A minority alter the protein's function, and a few of those are advantageous, which is one source of the variation on which natural selection acts.

The standard example, stated precisely: in sickle-cell anaemia a single base substitution in the gene for the β-globin chain of haemoglobin changes one triplet, one amino acid changes from glutamic acid to valine, and the altered haemoglobin polymerises at low oxygen concentrations and distorts the red blood cell. The gene sits on chromosome 11, so that is one base out of about 135 million on that chromosome.

Extracting and precipitating DNA

DNA can be extracted from tissue in a standard bench practical using ordinary reagents, and each step depends on a property of the molecule described above. Kiwi fruit, strawberries, peas, onion and cheek cells are all suitable sources.

Mash the tissue first, which breaks open the plant cell walls, then mix it with a warm solution of salt and detergent and leave it for a few minutes. The two reagents do different jobs. The detergent disrupts the phospholipid bilayers of the cell surface membrane and the nuclear envelope, because membranes are lipid and dissolving lipid is what a detergent does; that releases the DNA into solution. The sodium ions of the salt are attracted to the negatively charged phosphate groups of the sugar-phosphate backbone and shield them, so the DNA molecules stop repelling one another and can later clump together. Filter the mixture to take out the debris of walls and organelles.

Next add a protease. In a eukaryotic nucleus the DNA is wound around histone proteins, and the extract is full of other protein besides; the protease hydrolyses them, leaving the DNA free. Then tilt the tube and pour ice-cold ethanol slowly down the side, so that it forms a separate layer floating on the extract rather than mixing in. DNA is insoluble in cold ethanol, so it comes out of solution at the boundary between the layers as a tangle of white fibres, which can be lifted out by winding them slowly onto a glass rod. That lifting-out is called spooling, and it only works because each molecule is so enormously long. The cold matters twice over: DNA is less soluble in cold ethanol than warm, and low temperature slows any enzymes in the extract that would otherwise be cutting the molecule up.

Each reagent has a distinct function, and each follows from the structure of the material. Detergent disrupts membranes because membranes are phospholipid; salt is required because the backbone carries a negative charge at every phosphate; protease is required because eukaryotic DNA is packaged with histones; and the fibres are visible without magnification because the molecules are millions of base pairs long.

DNA precipitation

The recovery of DNA from a filtered tissue extract as visible fibres, by layering ice-cold ethanol on top; the DNA, insoluble in cold ethanol, forms at the boundary and is spooled onto a rod.