DNA replication is the process by which DNA is copied, usually before a cell divides, and this process requires the action of many different proteins to ensure replication occurs accurately and quickly.
DNA replication is semiconservative, meaning that each strand in the DNA double helix acts as a template for the synthesis of a new, complementary strand. Replication always starts at specific locations on the DNA, which are called origins of replication and are recognized by their sequence. An enzyme called helicase unzips the DNA by breaking the hydrogen bonds between the nitrogenous base pairs. ATP hydrolysis is required for this process. Specialized proteins recognize the origin, bind to this site, and open up the DNA. As the DNA opens, two Y-shaped structures called replication forks are formed, together making up what’s called a replication bubble. The replication forks will move in opposite directions as replication proceeds.
One of the key molecules in DNA replication is the enzyme DNA polymerase. DNA polymerases are responsible for synthesizing DNA: they add free nucleotides one by one to the growing DNA chain, incorporating only those that are complementary to the template. DNA polymerase is able to add nucleotides only in the 5′ to 3′ direction.
A consequence of the structure of nucleotides is that a polynucleotide chain has directionality – that is, it has two ends that are different from each other. You can see this in the figure below. At the 5’ end, or beginning, of the chain, the 5’ phosphate group of the first nucleotide in the chain sticks out. At the other end, called the 3’ end, the 3’ hydroxyl of the last nucleotide added to the chain is exposed. As new nucleotides are added to a strand of DNA or RNA, the strand grows at its 3’ end, with the 5′ phosphate of an incoming nucleotide attaching to the hydroxyl group at the 3’ end of the chain. This makes a chain with each sugar joined to its neighbors by a set of bonds called a phosphodiester linkage.
Therefore, DNA polymerases require a free 3′-OH group to which it can add free nucleotides by forming a phosphodiester bond between the 3′-OH end and the 5′ phosphate of the next nucleotide. This means that it cannot add nucleotides if a free 3′-OH group is not available. This ensures that there is specific coupling of free nucleic acids (nucleotides) during DNA replication. Another enzyme, RNA primase, synthesizes an RNA primer that is about five to ten nucleotides long and complementary to the DNA, priming DNA synthesis. This primer provides the free 3′-OH end to start replication, as you can see illustrated below:
DNA replication requires other enzymes in addition to DNA polymerase, including DNA primase, DNA helicase, DNA ligase, and topoisomerase.
• DNA replication is semiconservative.
• Helicase separates the double-stranded DNA helix to form a replication fork at the origin of replication where DNA replication begins.
• Replication forks extend bi-directionally as replication continues.
• DNA polymerase can add nucleotides only in 5′ to 3′ direction, requires a template, and can only add a new nucleotide where a free 3′-OH group is available. This ensures the selective coupling of free nucleotides.
• The directionality of DNA polymerase and the fact that the DNA double helix is always anti-parallel means that the two DNA strands are copied in different ways.
• Replication of the leading strand is continuous while replication of the lagging strand is discontinuous.
Semiconservative: Describes how newly formed double-stranded DNA molecules after replication contain one old strand and one new strand of DNA.
Origin of replication: A particular sequence in a genome at which replication is initiated.
Helicase: An enzyme that unzips the DNA helix by breaking hydrogen bonds between nucleotide base pairs.
ATP hydrolysis: The release of chemical energy, stored in a high-energy phosphoanhydride bond in ATP, by breaking the bond.
Replication fork: A Y-shaped structure that forms as the double-stranded DNA helix is separated by helicase.
Replication bubble: The structure formed as helicase enzymes separate the DNA strands at origins of replication.
DNA polymerase: An enzyme that mediates DNA replication by adding free nucleotides in the 5′ to 3′ direction.
Nucleotides (nucleic acids): A molecule that contains a nucleoside linked to a phosphate group; the building blocks of DNA.
Phosphodiester linkage: The bond formed between the 3′-OH end of one nucleotide and the 5′ phosphate of the next nucleotide.
Leading strand: The template strand of the DNA double helix that is replicated continuously in the 3′ to 5′ direction and is oriented in the same direction as the replication fork.
Okazaki fragments: Short sequences of DNA that are made discontinuously and later ligated together to create the lagging strand.
Lagging strand: One of the two newly synthesized DNA strands that must be synthesized in the opposite direction from the replication fork.
DNA Primase: An enzyme that synthesizes RNA primers complementary to the DNA strand.
DNA ligase: An enzyme that seals gaps between Okazaki fragments on the lagging strand.
Topoisomerase: An enzyme that functions ahead of the replication fork to prevent supercoiling of the DNA by introducing breaks and then sealing them.