How DNA Replication Works (Video)

0:10 The question after the double helix

First, we will look at the question the double helix raised. In 1953 Watson and Crick proposed the double helix, in which each base on one strand pairs with a specific base on the other. They noted that this pairing suggests a copying mechanism for the genetic material. Three models could produce the copy. Semiconservative replication keeps one old strand in each new double helix and adds one new strand. Conservative replication leaves the old double helix intact and builds an entirely new copy. Dispersive replication mixes old and new DNA in pieces along both strands. Matthew Meselson and Franklin Stahl tested the three models in 1958. They grew Escherichia coli on heavy nitrogen, nitrogen fifteen, then moved the cells to light nitrogen, nitrogen fourteen. They separated the DNA by density in a caesium chloride gradient. After one generation, all the DNA formed a single band of intermediate density, and that result rules out the conservative model. After two generations, the DNA formed two bands, one intermediate and one light. The dispersive model cannot produce a light band, so semiconservative replication was the only model left. I request that you watch the video till the end because then we can make more of these videos as the algorithm recommends it to more people because of increased retention time. And that really helps us a lot.

1:36 What the enzyme can and cannot do

Second, we will see what the enzyme can and cannot do. To follow how the cell makes the copy, you need two facts about the structure. The two strands are antiparallel, so one runs from the five prime end to the three prime end and the other runs from three prime to five prime. The bases pair adenine with thymine and guanine with cytosine. DNA polymerase reads the template strand and adds each new nucleotide to the free three prime hydroxyl group of the growing strand. A new strand therefore grows in the five prime to three prime direction. DNA polymerase cannot start a new strand on its own, because it can only extend a strand that already exists. Each new strand begins with a short primer.

2:22 The origin and the replication fork

Third, we will follow the origin and the replication fork. With these rules in place, you can follow the copy from its starting point. Replication begins at specific sequences, the origins of replication. The circular chromosome of Escherichia coli has a single origin, and a human cell uses tens of thousands of origins. Helicase separates the two strands by breaking the hydrogen bonds between the bases, which opens a Y-shaped replication fork. Single-strand binding proteins coat the exposed strands so that they do not pair again. Unwinding over-twists the DNA ahead of the fork, and topoisomerase cuts and rejoins the DNA to relieve that strain. In bacteria, DNA gyrase carries out this step. From each origin, two forks move in opposite directions, so replication is bidirectional.

3:14 Primers and the polymerase

The fourth part covers primers and the polymerase. Once the strands are open, the polymerase still needs a primer to start from. Primase is an RNA polymerase, and it synthesizes a short RNA primer of about ten nucleotides on the template. DNA polymerase III, the main replicative polymerase of Escherichia coli, extends the primer and adds about one thousand nucleotides per second. A ring-shaped sliding clamp holds the polymerase on the DNA so that the enzyme does not fall off.

3:46 Leading and lagging strands

Fifth, we will compare the leading and lagging strands. The strands are antiparallel, and DNA polymerase works only in the five prime to three prime direction, so the cell makes the two new strands differently at the same fork. The polymerase makes the leading strand continuously, in the direction the fork moves, from a single primer. If you follow the lagging strand, you see it grow away from the fork as a series of separate pieces, the Okazaki fragments. Each fragment starts from its own primer. Okazaki fragments run about one thousand to two thousand nucleotides in bacteria and about one hundred to two hundred in eukaryotes. In Escherichia coli, DNA polymerase I removes the RNA primers and fills the gaps with DNA. Breaks remain in the sugar-phosphate backbone at the joins between fragments. DNA ligase seals these nicks and joins the fragments into one continuous strand.

4:44 How accurate the copy is

The last part shows how accurate the copy is. A polymerase this fast also makes mistakes. Base pairing alone would allow roughly one error in a hundred thousand nucleotides. The replicative polymerase also proofreads each new base pair. When it detects a mismatch, its three prime to five prime exonuclease activity removes the wrong nucleotide. Proofreading improves accuracy to about one error in ten million nucleotides. After replication, mismatch repair corrects most remaining errors, so the final error rate is about one in a billion nucleotides. Here is what you learned in this video. The Meselson and Stahl experiment showed that replication is semiconservative, with one old strand kept in each new double helix. At each origin, helicase separates the strands and opens two replication forks. DNA polymerase works only in the five prime to three prime direction, so the leading strand grows continuously and the lagging strand grows as Okazaki fragments that DNA ligase joins. Here is a question for you to test what you learned. After two generations in light nitrogen, why could the dispersive model not explain the bands that Meselson and Stahl observed? Pause the video and work it out. The answer follows. The dispersive model predicts a single band that moves toward light density, but Meselson and Stahl saw two separate bands, one hybrid and one light, and only the semiconservative model explains that pattern.