DNA ligase makes recombinant DNA technology possible.
In this lesson, you will learn how new versions of genes can be designed for experiments in novel host organisms using DNA ligase.
The goal of genetic engineering is changing the genetic makeup of an organism. Recall that we previously learned that a restriction enzyme is an enzyme that can cut DNA and that these enzymes are commonly used in genetic engineering experiments. Scientists can use restriction enzymes to excise a specific gene from a genome and add it to a DNA plasmid to create a completely new recombinant version of the gene.But, restriction enzymes just provide half of the story, right? To actually create recombinant DNA, pieces of DNA must be pasted together. That’s where the enzyme DNA ligase comes in.
Review of DNA Ligase in Replication
If you recall, nature encountered a similar challenge to the one we’re facing as scientists.
During DNA replication, a cell makes an exact copy of each DNA molecule. However, because DNA polymerase (the enzyme that builds new DNA molecules) can only function in one direction with respect to DNA, a cell must treat each side of the replication fork slightly differently. At the leading strand, DNA polymerase can add nucleotides to build the new DNA molecule without stopping.In contrast, as the replication fork opens, discontinuous fragments of DNA are built at the lagging strand. These fragments are then glued, or ligated together, by DNA ligase.
Mechanism of DNA Ligase
Now, you may wonder why DNA ligase is even necessary. I mean, the hydrogen bonds are holding the two molecules together. That should be good enough, right? Recall, though, that hydrogen bonds are constantly broken and reformed during such cellular processes as replication and transcription.Consider the structure of a DNA nucleotide. A nucleotide consists of a nitrogenous base, a sugar, and a phosphate.
So far, we’ve been considering the hydrogen bonds that hold the complementary bases together in a DNA molecule. Now let’s consider the bonds that hold each DNA backbone together.Recall that the backbone of each DNA strand is composed of the sugars and phosphates of the individual nucleotides linked by a covalent bond called a phosphodiester bond. To generate the DNA fragments, the restriction enzymes broke the phosphodiester bond holding the sugar and phosphate together. Because a covalent bond is a high-energy bond, an enzyme is required to make forming the bond energetically possible. DNA ligase serves this role.
In our experiment, it will glue the human insulin gene into a bacterial DNA plasmid, thus forming a recombinant DNA molecule that will soon be able to produce human insulin. Note that we have only considered a specific genetic engineering experiment that utilizes restriction enzymes that generate sticky ends. However, while not all forms of genetic engineering require these specific enzymes, virtually all recombinant DNA experiments require the use of DNA ligase to create a recombinant plasmid for storage or amplification.
Restriction enzymes are enzymes that can cut DNA.
You can think of restriction enzymes as molecular scissors. DNA ligase is an enzyme that can join two DNA molecules. You can think of DNA ligase as molecular glue. DNA ligase joins two DNA molecules together by forming a phosphodiester bond between the two molecules.
At the end of this lesson, you’ll be able to define restriction enzymes and DNA ligase and explain their functions in engineering recombinant DNA.