Daily Newsletter

September 5, 2014 - DNA Replication

Textbooks have a tendency to make replication one of the most complext topics covered. With a tendency to throw all the current research and understanding at students, they rarely take a step back and try to explain it. This newsletters has two goals: 1) to help biology students understand DNA replication, and 2) to show you want is expected from a Biology Freshman/Sophmore.

Central Dogma of Biology

Before we get into replication, let's take a step back and look at the three core genetic processes, a.k.a., the Central Dogma. The central dogma describes the flow of genetic information in a cell. The core idea is INFORMATION. You may recall some of our early discussion on DNA, and about base complimentarity and the directionality of the molecule. This will become important rather quickly, but for just this moment, I want you to concentrate on the fact that DNA carries information. Information on how to build RNA and Proteins, both of which will produce the phenotype (expression) of the cell. For this reason, DNA, RNA and Proteins are considered Informational Macromolecules. This means that the sequence of monomers contains infomation, e.g., instructions on how to build RNA and Proteins. Since it is critical, the sequence of nucleotides carries information.
As you can see from the diagram of central metabolism shown above, there are three processes: Replication, Transcription and Translation. Think about those words. They are words used in reference to languages and documents. When you replicate a docuement, you want to ensure that you are getting a faithful (or even exact) copy of the original.

When you transcribe, you are moving from one medium, e.g., spoken word, to another medium, e.g., text. If you watch news shows, they will tell you that transcripts of the show are available. Court reports make transcripts of the trial. You are taking the language from one medium (in our case DNA) to another medium (RNA). The language is still the same (i.e., nucleic acid lanugae), just in a different form. Does the transcript have to be 100% correct? You want it to be, but it is not as exacting as a replication.
Translation is where you change languages. Unless you're fluent in another language, you will need somoene to help translate, or at least a good translation dictionary. Now you are moving the context from one language (nucleic acid) to another language (amino acids).

All of these processes rely on one key feature of nucleic acids: BASE COMPLIMENTARITY. In DNA: A complements T, and G complements C. In RNA: U complements A, and G still complements C.

Replication

In replication our goal is to take one molecule of DNA and make two daughter molecules of DNA that are identical to the first. Even the best replication processes can produce errors, but our goal is to be error free. This takes precision! As DNA is long, we also need this to be a fast process.

The enzyme that is ultimately responsible for replication is DNA Polymerase. [NOTE: there are multiple types of DNA Polymerase, but for now you just need to understand the core concept common to the DNA Polymerase family.] DNA polymerase is only one component of the Replication Complex, which is a complex association of proteins needed to successfully complete the replication event. Your goal at this time is to concentrate on DNA polymerase; we will talk about some of the other components later.

DNA is a double stranded molecule, in which the strands are anti-parallel. This means that one strand starts at the 5' end → 3' end, while the the other strand is revered. This can be seen in the image to the right. A common way of saying this is that we read DNA in the 3' → 5' direction. Why is this important? DNA Polymerase can only read DNA in the 3' → 5' direction, and can only build a new strand in the 5' → 3' direction. Before we go on, let's look another time at how deoxyribonucleotides are polymerized.

To the left you will see a generic image showing DNA Polymerase adding nucleotides to a growing DNA strand. Look carefully: A deoxyribonucleotide triphosphate (dTTP) is being added. The 5' phosphate of the new nucleotide is what will be used to form the phosphodiester bond. DNA Polymerase requires a free 3' end on which to grow the new DNA strand. To the right is another image that will help you with this concept. Phosphodiester Bond Formation

At this point, the critical thing to remember is that DNA Polymerase will need a free 3' end on which to add a new nucleotide.
This requirement to build in only one direction (5' → 3') creates a problem for the DNA process: the two strands read in opposite directions, and each must be replicated. It was noticed that one strand appears to replicate continuously, while the opposite strand appears to replicate discontinuously.

The original strand that reads 5' → 3' can be used by DNA polymerase to continuously produce the new 5' → 3' strand (the antiparallel complement to the original strand). We refer to the continuous synthesis strand as the Leading Strand. The other original strand, which read 3' → 5' cannot be copied continuously. A section will have to be exposed, replicated, and then another section exposed. This strand is constructed discontinuously, and is reffered to as the Lagging Strand. DNA Replication

The image above shows the leading and lagging strand. Notice that the leading strand is replicating toward the Replication Fork(where the original strands seperate). As more DNA unwinds and opens, DNA synthesis continues down the leading strand.
The lagging strand though has to build in a start-stop action, producing Okazaki fragments. These fragments have to be sealed (phosphodiester bonds) together before DNA can rewind into the α-helix.

In the image above, you will notice a number of enzymes on the lagging strand. These enzymes are required for the initiation of DNA polymerazation, and then sealing the fragments. Looking at the image, you will see an enzyme called Primase, and a structure in red known as a Primer. Another restriction on DNA polymerase is that it must have a free 3' end from which to start building. DNA Polymerase is prevented from building a DNA strand from nothing. Something (a primer) must be in place upon which DNA polymerase can build. The Primer is constructed from RNA, and is a temporary scaffold upon which DNA polymerase can start working.

Eventually the primer will need to be moved. This is where you need to learn a little more about DNA polymerase. There are multiple DNA polymerases in eukaryotic systems. The general work horse of replication is DNA Polymerase III (DNA pol III), which is used to make long strands of DNA. DNA Polymerase I (DNA pol I) is used to replace primers (it is also used in repair functions). Even after the RNA primer is replaced with DNA, there is still a gap between fragments. Ligase is the enzyme used to create a phosphodiester bond between fragments, thus sealing the new sugar-phosphate backbone of the synthesized strand.

The result of DNA replication is that one molecule one DNA was used to create two new molecules of DNA. The two strands of the original DNA molecule became the template from which to build new complimentary strands of DNA. This is referred to as semi-conservative replication, as each new molecule has one strand from the original molecule, and one freshly synthesized complimentary strand.