DNA Structure and Replication

07. DNA Structure and Replication

This chapter covers how DNA came to be recognized as the genetic material, the three dimensional architecture of the double helix, and the machinery that copies chromosomes. Replication is presented first in bacteria, where the enzymes are best characterized, then compared with the eukaryotic system.

Evidence That DNA Is the Genetic Material

In 1928 Frederick Griffith studied Streptococcus pneumoniae: the smooth (S) form kills mice because it has a capsule, the rough (R) form not. Mice given live R or heat killed S survived, while a mixture of heat killed S and live R killed them and yielded live S bacteria. Something from the dead S cells had converted R cells into the killer form, a change he called transformation; he named the agent the transforming principle.

In 1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty purified the principle: protease and ribonuclease left it intact, but deoxyribonuclease destroyed it, so it was DNA. In 1952 Alfred Hershey and Martha Chase confirmed this with the bacteriophage T2, which injects its genetic material into bacteria. One batch was labeled with radioactive phosphorus (P-32), marking only DNA, and another with radioactive sulfur (S-35), marking only protein. Nearly all the P-32 entered the bacteria and the S-35 stayed in the empty coats: DNA directs the production of new phage.

ELI-10

Two chefs each claim to keep their cake recipe in a different place: one on paper, one on metal. Dip the paper in a chemical that dissolves paper, or the metal in one that dissolves metal: if the paper recipe was real, destroying the paper destroys the recipe and the cake fails. Hershey and Chase used this logic to show that DNA, not protein, carries the instructions for making new viruses.

Common Mistake: Students often credit Griffith with proving that DNA is the genetic material. Griffith only showed that a transforming principle exists; Avery, MacLeod, and McCarty identified it as DNA, and Hershey and Chase confirmed it in 1952.

The Watson Crick Model

In 1953 James Watson and Francis Crick proposed the double helix model, built on Chargaff's rules (A equals T and G equals C) and the X ray diffraction of Rosalind Franklin and Maurice Wilkins. Two chains wind around a common axis, backbones outside and bases inward, paired by hydrogen bonds: A with T and G with C. The helix is right handed, 2.0 nanometers in diameter, rising 0.34 nanometers per base pair, with one turn per 10 base pairs. The surface divides unequally into the major groove and the minor groove.

ELI-10

Think of a spiral staircase with the handrails outside and the steps in the middle. Each step is two halves that fit together, and only correct pairs snap, so A always clicks into T and G into C. That is how the two sides of DNA line up.

A B and Z Forms

DNA can adopt several conformations. B DNA, the standard cellular form, is right handed with about 10 base pairs per turn. A DNA is right handed but wider and more compressed, about 11 base pairs per turn, favored when DNA is dehydrated or the duplex contains RNA. Z DNA is left handed with a zigzag backbone, about 12 base pairs per turn, favored in alternating GC runs. B DNA is the resting form; A and Z appear where the helix is unwound or packed.

ELI-10

A garden hose can be coiled in different ways, and the same hose is still a hose. Wind it one way and you get a right handed spiral, loosely and it gets fatter, twisted the other way and it becomes left handed. DNA does the same: B is the usual coil, and A and Z are alternative coils for different situations.

Antiparallel Strands and Base Pairing Chemistry

Nucleotides are joined by phosphodiester bonds from the 5' phosphate of one nucleotide to the 3' hydroxyl of the next, giving each strand a 5' end and a 3' end. In the helix the strands run opposite ways, called antiparallel: where one runs 5' to 3', the other runs 3' to 5'. The purines (adenine, guanine) have two rings; the pyrimidines (thymine, cytosine) have one. A purine always pairs with a pyrimidine, keeping every base pair the same width. Adenine pairs with thymine by two hydrogen bonds, guanine with cytosine by three. If the coding strand reads 5' ATG GCA TAA 3', the template strand reads 3' TAC CGT ATT 5'.

ELI-10

Think of a zipper that must close from the top and the bottom at the same time. One side has teeth pointing one way and the other side matching teeth pointing the opposite way, and each tooth snaps with exactly one partner. That is why the strands run opposite ways and each base has one correct partner.

Common Mistake: Asked for a complement, students often write the paired bases in the same direction, for example 5' TAC CGT ATT 3' for 5' ATG GCA TAA 3'. The sequence is right, but the direction is wrong: it must be written 3' TAC CGT ATT 5'.

Semiconservative Replication and the Meselson Stahl Experiment

In conservative replication the parental helix stays intact and a new helix is built; in semiconservative replication each daughter gets one old and one new strand; in dispersive replication old and new material is interspersed on both strands. In 1958 Matthew Meselson and Stanley Stahl distinguished the models with density labeling. They grew E. coli in heavy nitrogen (N-15) until all DNA was heavy, then shifted the cells to N-14 and sampled each generation. In a cesium chloride gradient the initial DNA formed one heavy band; after one generation, a single intermediate band, as semiconservative replication predicts and conservative replication cannot. After two generations half the DNA was intermediate and half light, which dispersive replication cannot produce. The intermediate to light ratio after two generations is 1:1 (sums to 2), so replication is semiconservative.

ELI-10

Suppose a notebook page is made by pasting a white page onto a yellow page. To copy it, tear the pages apart, give one half to each copy maker, and each pastes a fresh white page onto the yellow half. Every new page keeps one yellow half; after another round the yellow halves sit in only two pages.

Origin and Replication Fork

Replication starts at a defined sequence, the origin of replication. In E. coli this is oriC, where the DnaA initiator protein, encoded by the dnaA gene, binds and initiates replication. Two replication forks move in opposite directions around the circular chromosome, so replication is bidirectional; the partially replicated circle resembles the Greek letter theta, hence theta replication. Each fork carries the replisome, a multienzyme complex. Bacteria copy the whole chromosome from one origin; eukaryotes use many origins, and each stretch copied from one is a replicon.

ELI-10

Imagine a long loop of road that needs repaving, and two crews start at the same point, one going clockwise and the other counterclockwise. They meet on the far side of the loop, so the whole loop is paved in half the time; a eukaryotic chromosome is like a much longer road with many crews starting along it at once. Each crew is a replication fork, and their starting point is the origin.

Enzymes at the Replication Fork

The DnaB helicase (dnaB) unwinds the duplex at the fork. Single strand binding (SSB) proteins coat the single stranded DNA so the templates cannot rejoin. DNA gyrase, a topoisomerase of the GyrA and GyrB subunits encoded by gyrA and gyrB, introduces negative supercoils that relieve the strain. DNA polymerases can only add to an existing 3' hydroxyl, so DnaG primase (dnaG) makes the RNA primers that start each chain. DNA polymerase III, whose alpha subunit is encoded by dnaE, is the main replicative polymerase. DNA polymerase I, encoded by polA, removes the RNA primers and fills the gaps. DNA ligase, encoded by ligA, seals the nicks.

ELI-10

Copying a long book is a job for several specialists: one unzips the book, another holds the pages open, and a third marks fresh starting spots. The main writer copies, a second fixes rough spots, and a glue worker joins the pages. Each worker has one specialty, and all must work in order.

Common Mistake: DNA polymerase I is not the main replicative polymerase. DNA polymerase III does the bulk of chain extension; DNA polymerase I removes the RNA primers and fills the gaps.

Leading and Lagging Strand Synthesis

Every new strand is synthesized 5' to 3', and every template is read 3' to 5'. Because the fork unwinds in one direction, it moves toward the 3' end of one template and the 5' end of the other. The leading strand is synthesized continuously, in the direction the fork moves, from a single primer. The lagging strand is synthesized discontinuously, away from the fork, in short pieces from fresh primers.

ELI-10

Imagine copying a poem from a moving billboard while standing still. If the words move toward you, copy the whole poem in one steady pass; if they move away, you can only catch a short stretch at a time. The pieces are taped together afterward, and both copies were written the same way.

Common Mistake: Some students claim the lagging strand is synthesized 3' to 5'. Both new strands are synthesized 5' to 3'. The lagging strand is built in short pieces pointing back toward the origin.

Okazaki Fragments

The short pieces of lagging strand DNA are the Okazaki fragments, named for Reiji Okazaki. Each begins with an RNA primer and continues with DNA until it meets the primer of the fragment ahead. Fragments are roughly 1,000 to 2,000 nucleotides in E. coli and 100 to 200 in eukaryotes. DNA polymerase I removes each primer and fills the gap; DNA ligase seals the nick.

ELI-10

If a wall must be built while the blueprint keeps sliding out of reach, the builder works in sections: she lays one row of bricks, waits for the next section to come close, then lays the next row with a temporary marker post. At the end, someone replaces the marker posts with real bricks and glues the rows together. The wall looks seamless, but it was built from separate sections.

Proofreading

DNA polymerase III copies quickly but still errs, so it carries an editing function called proofreading. When it inserts a nucleotide it checks the new base pair; if wrong, a 3' to 5' exonuclease removes it before synthesis resumes. The error rate is about one mistake per 10^5 nucleotides without proofreading and about one per 10^7 with it.

ELI-10

When you copy homework, you write one letter and immediately check it before writing the next. If a letter is wrong, you erase it at once and rewrite it. Catching the error while still on that letter is far better than rereading the whole page at the end. The polymerase does the same check after every nucleotide.

Common Mistake: Students sometimes think the polymerase fixes mistakes only after the strand is finished. Proofreading works during synthesis, removing a mispaired nucleotide from the 3' end the moment it is detected.

Telomeres and Telomerase

Linear chromosomes face an end replication problem: the final RNA primer on the lagging strand sits at the very end of the template, and once removed, nothing upstream exists to extend, so the end shortens each division. The countermeasure is the telomere, a repeated sequence at each chromosome end; the human telomeric repeat is 5' TTAGGG 3'. Telomerase solves the problem: its RNA component, encoded by TERC (at 3q26.2), carries the template for the repeats, and its catalytic subunit, the telomerase reverse transcriptase encoded by TERT (at 5p15.33), elongates them. Telomerase is active in germ line and stem cells, largely off in most somatic cells.

ELI-10

The plastic tip at the end of a shoelace keeps the lace from unraveling. Every time the lace gets pulled hard, the tip wears down a little, and once it is gone the lace starts to fray. A special machine can attach a new tip, using a tiny pattern to make the same shape every time. Telomerase is that machine for chromosome ends, and the pattern is stored in its own RNA piece.

Eukaryotic versus Prokaryotic Replication

Bacteria carry one circular chromosome with a single origin; eukaryotes have many linear chromosomes with many origins each. Bacterial forks move at about 1,000 nucleotides per second, an order of magnitude faster than eukaryotic forks at about 50 per second. Eukaryotic DNA is wrapped around histones into nucleosomes that must be assembled on both daughters, and linear chromosomes need telomerase.

ELI-10

Copying a big textbook can be done in two ways: a single copying machine working through the whole book, or many machines each copying a short section at once. The second way finishes much faster but needs many machines. The copies must be fitted together at the seams.

Worked Example Meselson Stahl Generations

Problem: In a Meselson and Stahl experiment, cells grown in N-15 are shifted to N-14 and replicate for five generations. What fraction of the DNA is hybrid, with one N-15 and one N-14 strand?

Given: All starting DNA is heavy; replication is semiconservative; each generation doubles the number of molecules.

Plan: Count the molecules after five generations, then count how many contain one of the two original heavy strands.

Solution: Total molecules = 2 x 2 x 2 x 2 x 2 = 32. The two original heavy strands are conserved, one in each of two hybrids, so fraction hybrid = 2 / 32 = 1 / 16 = 0.0625.

Answer: 1/16, or 6.25 percent, is hybrid after five generations.

Worked Example Okazaki Fragment Count

Problem: A 4.8 Mb circular bacterial chromosome replicates bidirectionally from one origin, and lagging strand Okazaki fragments average 1,000 nucleotides. How many fragments are made in one round?

Given: 4.8 Mb = 4,800,000 base pairs; two forks; average fragment length 1,000 nucleotides.

Plan: Lagging strand synthesis across both forks equals the full chromosome length; divide by the fragment length.

Solution: 4,800,000 / 2 = 2,400,000 base pairs per fork, and 2 x 2,400,000 = 4,800,000 nucleotides of lagging strand synthesis in total. Fragments = 4,800,000 / 1,000 = 4,800.

Answer: 4,800 Okazaki fragments are made per round of replication.

High-Yield:

• Both new strands are synthesized 5' to 3'; the template is always read 3' to 5'.
• DNA polymerase III is the main replicative polymerase; DNA polymerase I removes the RNA primers, and DNA ligase seals the nicks.
• The leading strand is continuous with one primer; the lagging strand is made of Okazaki fragments with many primers.
• In E. coli, DnaA initiates at the origin, DnaB unwinds the duplex, DnaG makes the primers, and DNA gyrase relieves the supercoiling.
• Telomerase, with its RNA template TERC and its catalytic subunit TERT, maintains the ends of linear eukaryotic chromosomes.

Quick Review

• Griffith showed transformation; Avery, MacLeod, and McCarty identified the transforming principle as DNA; Hershey and Chase confirmed it with phage T2.
• The Watson Crick double helix is right handed and antiparallel, with A pairing to T and G to C.
• Meselson and Stahl density labeling showed replication is semiconservative.
• DNA polymerase III extends both strands 5' to 3'; the lagging strand is built from Okazaki fragments.
• DNA polymerase I removes the RNA primers, and DNA ligase seals the nicks.
• Proofreading by the 3' to 5' exonuclease lowers the error rate to about one mistake per 10^7 nucleotides.
• Telomerase uses TERC as the template and TERT as the catalytic subunit.