The machinery of replication · Biology Class 11–12 | StemPy Lab
Course 23.2·Lesson 2 of 3
The machinery of replication
Copying starts at an origin of replication and goes both ways, which makes a Y-shaped replication fork. DNA polymerase uses a DNA template and the four deoxynucleoside triphosphates and adds nucleotides only in the 5 prime to 3 prime direction. So the leading strand is made in one piece, and the lagging strand is made as Okazaki fragments that DNA ligase joins.
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Where does the copying start?
A long DNA does not start to copy at any place. It starts at a fixed place, the origin of replication, written ori. A piece of DNA can be copied only if it has an ori. A bacterial chromosome has one origin. A chromosome of a eukaryote has many.
At the origin, the two strands come apart, and the copying goes in both directions.
A circular bacterial chromosome drawn as a grey ring. At the top an orange dot marks the origin of replication, ori. From it a red arc of new DNA runs inside the ring in both directions. At each end of the arc a black arrow points along the ring, away from the origin, to show the two replication forks moving in opposite directions. A caption says that two forks start at the origin and move in opposite directions, so the copying is bidirectional.
The place where the strands are coming apart is the replication fork. It looks like the letter Y. In a bacterial chromosome two forks start at the origin and move round the ring in opposite directions.
How fast is it?
NCERT gives the chromosome of E. coli, 4.6×106 base pairs, and says that it is copied in about 18 minutes. This needs about 2000 base pairs a second. In a human cell the DNA is 6.6×109 base pairs, and the S phase takes about 8 hours. A human cell makes up for this size with a very large number of origins, so that many forks work at once.
You should find that the time falls in proportion to the number of origins, and rises in proportion to the size of the DNA.
The enzyme
The main enzyme is DNA-dependent DNA polymerase. It uses a DNA template to join deoxynucleotides into a new strand, following the rules of base pairing.
The building blocks are the deoxynucleoside triphosphates, dATP, dGTP, dCTP and dTTP.
Each one has three phosphates. When it is joined to the strand, two phosphates are broken off. This gives the energy for the joining. So the same molecules are the substrate and the energy source.
The polymerase can add a nucleotide only to the 3′ end of a strand. So a new strand always grows from its 5′ end towards its 3′ end. It can never grow the other way.
The leading and the lagging strand
The two template strands run in opposite directions, but the polymerase can work in one direction only.
A replication fork drawn sideways. The parent DNA is on the right, two straight lines, a blue one on top and a grey one below. To the left the strands separate into a Y. The blue top template runs from 5 prime on the right to 3 prime at the upper left. The grey bottom template runs from 3 prime on the right to 5 prime at the lower left. An arrow shows that the fork moves to the right. A red leading strand lies along the blue template and grows towards the fork, in one piece, with a DNA polymerase on it. Three short green pieces lie along the grey template, each one growing away from the fork, to the left, with a DNA polymerase on the newest. They are the Okazaki fragments of the lagging strand, and DNA ligase joins them.
On the template that is read from 3′ to 5′ towards the fork, the new strand grows towards the fork, as the fork opens. It is made in one piece. It is the leading strand, and its copying is continuous.
The other template runs the other way. The new strand can only grow away from the fork, so it must be made in short pieces, each started after more of the template is open. These are the Okazaki fragments. Their copying is discontinuous. The strand is the lagging strand.
The fragments are joined by the enzyme DNA ligase.
Okazaki fragments are about 1000 to 2000 nucleotides long in bacteria, and about 100 to 200 nucleotides long in eukaryotes.
What else is needed?
This part is not in the NEET syllabus, but A-Level and IB ask it.
Helicase unwinds the helix and opens the fork, by breaking the hydrogen bonds. It uses ATP.
Single-strand binding proteins hold the open strands apart. Topoisomerase (in bacteria, gyrase) cuts and joins the DNA to relieve the twisting ahead of the fork.
Primase makes a short piece of RNA, the primer, because a DNA polymerase cannot start a new strand. It can only extend one. In bacteria, DNA polymerase III does most of the copying, and DNA polymerase I replaces the primers with DNA.
The polymerase proofreads. It removes a wrong base that it has just added. After the checks, the DNA is copied with about one mistake in a billion bases.
Try it in Python
The program counts the Okazaki fragments and the joints for a stretch of DNA, and the time at one fork.
Practise with new numbers
Common mistakes
Saying that the polymerase adds to the 5′ end. It adds to the 3′ end, and the new strand grows 5′ to 3′.
Saying that both strands are made in pieces. Only the lagging strand is made in fragments.
Forgetting that there are two forks at an origin. They work in two directions.
Mixing up the ligase and the polymerase. The polymerase adds nucleotides. The ligase joins the fragments.
Saying that the substrate is a deoxynucleotide monophosphate. It is a triphosphate, and this gives the energy.
Remember these
Replication starts at an ori and goes both ways, with a Y-shaped fork.
DNA-dependent DNA polymerase uses a template and dNTPs, and grows a strand 5′ to 3′.
Leading strand: continuous. Lagging strand: discontinuous, as Okazaki fragments joined by DNA ligase.
E. coli is copied in about 18 minutes, at about 2000 base pairs a second at a fork.
Check yourself
Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.
Why is only one strand of the new DNA made in pieces?
What is the job of DNA ligase?
Why are the deoxynucleoside triphosphates good as building blocks?
A chromosome has two origins, and four forks work at once. Why does the copying take less time?
How is a single origin enough for the chromosome of a bacterium?
References
NCERT (2023). Biology, Textbook for Class XII, chapter 5, Molecular Basis of Inheritance.
Clark M. A. et al. (2018). Biology 2e, OpenStax, chapter 14, sections 14.3 to 14.5, Basics of DNA Replication, DNA Replication in Prokaryotes and in Eukaryotes.
Alberts B. et al. (2022). Molecular Biology of the Cell, 7th edition, W. W. Norton, chapter 5, DNA Replication, Repair and Recombination.
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