8.2 DNA Replication
Key Takeaways
Replication is semiconservative: each daughter molecule has one parental strand and one new strand, the pattern supported by Meselson and Stahl.
Helicase unwinds the helix, primase lays down a short RNA primer, and DNA polymerase adds nucleotides only to a 3-prime hydroxyl.
New DNA is always synthesized 5-prime to 3-prime. The leading strand is continuous toward the fork. The lagging strand is made as Okazaki fragments, each still 5-prime to 3-prime.
DNA ligase joins fragments after the RNA primers are replaced. Polymerase proofreading removes a mismatch from the 3-prime end, but the chain still grows 5-prime to 3-prime.
Prokaryotes usually have one origin of replication and eukaryotes have many. The template 3'-AATGCT-5' is copied as 5'-TTACGA-3'.
8.2 DNA Replication
Semiconservative replication produces two daughter molecules, and each one contains one parental strand plus one newly built strand. Meselson and Stahl tested this with bacterial DNA that had been labeled in heavy nitrogen and was then copied in light nitrogen. After one generation the DNA was entirely intermediate in density, the result expected when every daughter molecule pairs one parental strand with one new strand. A second generation produced both intermediate DNA and fully light DNA. Conservative copying would have kept a fully heavy parental molecule. Dispersive copying would have left a single band that grew lighter, not a light band beside a hybrid band.
Opening the Fork
Copying starts at an origin of replication. Helicase unwinds the helix there, breaking the hydrogen bonds between bases. The covalent sugar-phosphate backbones stay intact, so each parent strand remains a continuous template. Single-strand binding proteins coat the separated strands so the bases do not pair again before they are copied. Ahead of the moving fork, unwinding overwinds the DNA that is still double-stranded. Topoisomerase cuts a backbone temporarily, lets the extra twist relax, and reseals the backbone. The opened zone, where the two templates diverge, is the replication fork.
Prokaryotes usually have a single origin. A circular bacterial chromosome can be copied by two forks that leave that origin and later meet on the far side. Eukaryotes have many origins on each long chromosome, so many forks work during one S phase.
Primers and a Polymerase That Only Adds to a 3-Prime End
DNA polymerase cannot start a new chain on a bare template. Primase, an RNA polymerase, synthesizes a short RNA primer first. The primer's 3-prime end has a free hydroxyl group, and that hydroxyl is the chemical handle the DNA polymerase needs. DNA polymerase then adds a deoxyribonucleotide only to a 3-prime hydroxyl. The new DNA strand therefore grows 5-prime to 3-prime, and it grows only in that direction. The polymerase reads the template from the template's 3-prime end toward its 5-prime end. That reading direction is the antiparallel consequence of the Watson-Crick structure. There is no synthesis reaction that builds a DNA strand by attaching new nucleotides onto a 5-prime end.
Leading Strand and Lagging Strand
The fork opens in one overall direction, but the two templates run opposite ways, so the two new strands cannot be made by the same uninterrupted motion. The leading strand is made continuously toward the fork. One primer can start it, and the polymerase keeps extending the same 3-prime end as helicase exposes more template. The lagging strand cannot be extended continuously in that same overall direction, because its new chain must still grow 5-prime to 3-prime. That growth points back toward the origin, away from the direction in which the fork is moving. Primase therefore sets down a new RNA primer each time a fresh stretch of lagging-strand template is exposed, and polymerase builds another short piece. Those pieces are Okazaki fragments. Every fragment is still synthesized 5-prime to 3-prime.
If the fork is opening to the right, the leading strand's new 3-prime end points that way and the polymerase can follow. The lagging strand's new 3-prime end points the other way, so each fragment is built away from the fork and needs its own primer. Fragments are the result of antiparallel strands. They are not evidence that a polymerase copies backward.
In Escherichia coli, DNA polymerase III carries out most of the elongation on both new strands. DNA polymerase I removes each RNA primer, using a 5-prime to 3-prime excision activity, and fills the resulting gap with DNA. DNA ligase seals the last nick by joining one fragment's backbone to the next, so the lagging strand becomes one continuous covalent chain. Eukaryotic forks use several DNA polymerases and shorter Okazaki fragments, but they still need primers, 5-prime to 3-prime addition, primer removal, and ligase.
| New strand | How synthesis proceeds | Direction of each new DNA chain |
|---|---|---|
| Leading | Continuous, following the opening fork, after one primer | 5-prime to 3-prime, toward the fork |
| Lagging | Okazaki fragments, each started with its own RNA primer | 5-prime to 3-prime on every fragment, away from the fork's overall movement |
Warning
DNA polymerase does not build a new strand in the 3-prime to 5-prime direction. When the template geometry seems to demand backward copying, the cell makes Okazaki fragments, and each fragment still grows 5-prime to 3-prime. Ligase joins them only after the RNA primers are replaced with DNA.
A Six-Base Copy
Use the template 3'-AATGCT-5'. Read it from its 3-prime end toward its 5-prime end, and write the new bases from 5-prime toward 3-prime. The first template adenine pairs with thymine. The next adenine pairs with thymine. The template thymine pairs with adenine. The template guanine pairs with cytosine. The template cytosine pairs with guanine. The final template thymine pairs with adenine. The product is 5'-TTACGA-3'.
A same-base copy such as 5'-AATGCT-3' repeats the template instead of complementing it. An RNA-style product such as 5'-UUACGA-3' is not the DNA strand, because DNA uses thymine in place of uracil. Writing the letters TTACGA and then claiming the polymerase grew them from the 3-prime end toward the 5-prime end contradicts the direction rule. The letters are right only when the new chain is 5'-TTACGA-3'.
Proofreading, and Why the Origin Count Differs
Proofreading happens at the growing end, immediately after a nucleotide is added. If the new base does not pair with the template, the polymerase removes that nucleotide from the 3-prime end and tries again. The removal step runs opposite the direction of chain growth, but it is editing, not a second mode of synthesis. After the correction, the next correct nucleotide is again added to a 3-prime hydroxyl, so the chain continues 5-prime to 3-prime. A mismatch that escapes proofreading can be copied later and become a permanent sequence change.
A eukaryotic chromosome is much longer than a typical bacterial chromosome, so many origins fire during S phase. Every origin still uses a primer, 5-prime to 3-prime extension, and ligase on the lagging strand. The worked copy does not change with the origin count: template 3'-AATGCT-5' becomes 5'-TTACGA-3', and the polymerase does not build that product 3-prime to 5-prime.
What did the Meselson and Stahl density results show about each daughter DNA molecule?
Its two strands are mixtures of parental pieces and new pieces scattered along their whole length.
Both of its strands are newly built, while the parental double helix remains fully heavy and intact.
DNA polymerase builds one daughter strand by adding nucleotides in the 3-prime to 5-prime direction.
It contains one parental strand and one newly made strand.
A template strand reads 3'-AATGCT-5'. Which new DNA strand does DNA polymerase produce?
3'-TTACGA-5', grown in the 3-prime to 5-prime direction
5'-UUACGA-3', with uracil in the DNA product
5'-AATGCT-3', a same-base copy in the same order
5'-TTACGA-3'
Why is the lagging strand made as Okazaki fragments?
DNA polymerase copies that template only by adding nucleotides in the 3-prime to 5-prime direction.
Helicase seals the fragments, and primase proofreads the bases after the fork has passed.
Each fragment is still synthesized 5-prime to 3-prime, while the lagging strand as a whole is extended by repeated starts as the fork opens.
Only eukaryotes have a lagging strand, because a eukaryotic chromosome has a single origin.
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