Introduction and Goals
Watson and Crick concluded their 1953 paper on the structure of DNA with the following statement:
"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
Figure 1. The structure of DNA. (Click to enlarge). Notice the base pairing rules - A always pairs with T and G always pairs with C.
The "specific pairing" they mentioned is the hydrogen-bonds between adenine and thymine, and guanine and cytosine (Fig. 1). The "copying mechanism" is DNA replication, a simple yet accurate series of steps by which DNA carries the instructions for its own reproduction.
This tutorial will cover the process of DNA replication. Remember, every time a cell divides - it must replicate its DNA. By the end of the tutorial you should have a basic understanding of:
- When DNA replication takes place
- Where DNA replication takes place
- How DNA replication takes place
- What happens if there is a mistake during DNA replication
- Compare and contrast the structures of DNA and RNA
- Diagram the process of DNA replication, including all of the enzymes and molecules that are necessary
- Understand the antiparallel structure of a DNA molecule and the implications this has for DNA replication
- Explain the different DNA repair mechanisms and the different possible types of mutations
Each time a cell divides, it must replicate its DNA. The replication of DNA takes place during the S-phase of the cell cycle (Fig. 2) and results in a chromosome that is comprised of two identical sister chromatids. DNA replication can be divided into several stages. The DNA double helix unwinds and nitrogenous bases (A,T,G or C) are added to each strand of the parent molecule (but only onto one end of each), resulting in two identical copies of the original parent strands.
Figure 2. The cell cycle. DNA replication occurs during the S-phase of interphase. (Click image to enlarge)
Figure 3. Semiconservative DNA Replication. (Click to enlarge). Each new molecule of DNA is comprised of one parental strand and one newly synthesized strand.
DNA replication is termed "semiconservative" replication because each newly formed molecule of DNA has one strand conserved from the parent molecule and one newly synthesized strand (Fig. 3).
This is an animation of an overview of DNA replication.
Along a strand of DNA, replication begins at numerous origins of replication. There are several enzymes involved in the process of DNA replication. Helicases unwind the DNA double helix, single-strand binding proteins keep the strands separate while primases initiate replication, and DNA polymerase adds nucleotides to the unwound parent molecule. While the fundamentals of replication are simple, there is a feature of DNA structure that makes things a bit more complicated; the strands have opposite chemical polarities. This can be hard to comprehend because the strands seem identical. However, a close inspection reveals that the H-bonding that occurs between bases is only achieved if the strands have opposite polarities. This arrangement of strands is antiparallel, with one strand designated the 3'-to-5' strand and the other the 5'-to-3' strand (Fig. 4).
Figure 4. DNA structure. Note that the 3' end of one strand is aligned with the 5' end of the complementary strand. (Click to enlarge).
DNA polymerase has an important limitation - it can only add nucleotides to the 3' end of the newly synthesized strand of DNA. Therefore, nucleotide addition is a smooth, continuous process along one of the strands (the leading strand) of DNA. The other strand (the lagging strand) has a discontinuous mode of replication because DNA polymerase can only work by starting from the replication fork(where DNA is unwinding) and progressing outward (until it runs into a previously synthesized fragment). An added wrinkle to the process on the lagging strand is created by the lack of a continuous new strand; DNA polymerase can only add nucleotides to an existing 3' nucleotide. How is this lagging strand started? Primase has the ability to synthesize a short primer made of a few nucleotides of RNA. DNA polymerase can then add DNA nucleotides to the end of this primer sequence and synthesize relatively short stretches of DNA known as Okazaki fragments. An additional enzyme (ligase) seals the fragments into a continuous strand of DNA. Figure 5 provides an overview of the enzymes involved, antiparallelism, and the overall direction of DNA replication. All of this takes place in the nucleus of eukaryotic cells (cells that have membrane-bound nuclei and organelles). Be sure that you study this figure carefully. You could see this figure unlabeled and be asked to name the enzymes and their functions on an exam.
Figure 5. An overview of DNA replication. (Click to enlarge)
This is an animation of the steps of DNA replication.
Errors during DNA Replication result in Mutations
In spite of the rules of base-pairing, sometimes mistakes are made during DNA replication. Mistakes occur about once in every 10,000 base pairs and can potentially be disastrous for an organism. There are various repair mechanisms that can fix these errors and, in the end, the observed error rate is very low (often less than one mistake/10 million bases). Mismatch repair occurs when DNA polymerase and other proofreading enzymes remove incorrectly paired nucleotides. Excision repair involves the removal of damaged nucleotides from a DNA molecule.
If these repair mechanisms are not successful, mutations occur. A mutation is a permanent change in an organism's DNA. If the mutation occurs in a reproductive cell, the mutation can be passed to future generations and potentially become established in a population (this is why mutations are the ultimate source of genetic variation in populations). The effect that a mutation has on an organism depends upon whether or not the mutation occurs in a gene and, if it does occur in a gene, how much it changes the resulting protein. Therefore, mutations can be harmful (e.g., sickle cell anemia, cystic fibrosis), beneficial (e.g., antibiotic resistance), or neutral (a neutral mutation occurs when an organism's DNA sequence changes but this change has no effect on the organism's phenotype).
An example of enzymes that help defend against mutations are specific DNA polymerases that fix errors that occur in DNA as a result of exposure to UV radiation (sunlight). Xeroderma pigmentosum (XP) is an autosomal recessive genetic condition that occurs as a result of a mutation to one of seven different DNA polymerases that repair UV-damaged DNA. The affected individual has a decreased ability to repair UV-induced mutations and the resulting damage is cumulative and irreversible. These individuals must stay away from sunlight and are sometimes referred to as "Children of the Night". People with XP begin developing skin cancers in childhood and life expectancy is significantly decreased.
This tutorial examined the process of DNA replication. This process is pivotal to life, so it will be important that you have a firm grasp on the basic aspects of DNA replication (i.e., to the level presented in this tutorial).
All cells that divide need to replicate their DNA so that each daughter cell contains a full complement of all the parent's genetic information. In the process of replication, the two strands are replicated with remarkable fidelity. To appreciate this process, keep a couple of things in mind. First, DNA is comprised of two antiparallel strands. The polarity of each strand is due to the manner in which the nucleotides are linked together. Second, DNA is synthesized in only one direction; 5' to 3'. Be sure that you understand that one strand is synthesized continuously, and that the other is synthesized discontinuously. Be sure that you understand why this is so, and be sure that you are familiar with the basic steps involved in this process.
After reading this tutorial, you should have a working knowledge of the following terms:
Case Study for DNA Replication
Myostatin is a protein that regulates the development and growth of muscle tissue in vertebrates. When laboratory mice have the myostatin gene experimentally “knocked out” they develop unusually large muscle mass (they have been called Schwarzenegger mice). As a result of these studies it was determined that myostatin limits the development of muscles and prevents them from growing too large.
In 1999, a child was born in Berlin, Germany with a mutation in both copies of the gene for myostatin production. The result is that he has twice the muscle mass and half the fat as most children his age.
A similar mutation is found in Belgian Blue cattle. These cattle are heavily muscled and provide more meat per animal than other cattle varieties. An analysis of the gene products of the normal and mutant alleles indicates that the mutant protein is shorter than the normal protein.
- If you were able to determine the sequence of nucleotides for both the normal and mutant alleles of the myostatin gene, what evidence would you look for to determine if the mutant protein was due to a deletion of nucleotides within the gene?
- How would you determine if the mutant gene was due to a stop codon in the wrong place?
Now that you have read this tutorial and worked through the case study, go to ANGEL and complete the tutorial practice problems to test your understanding. Questions? Either send your instructor a message through ANGEL or attend an online office hour (the times are posted on ANGEL).