Objectives

By the end of this lesson you should be able to:

  1. Describe Fredrick Griffiths ‘s experiment and the Hershey and Chase experiment which led to the discovery that DNA is the heritable factor
  2. Describe the structure of DNA
  3. Describe how Meselson and Stahl came to conclude that DNA replicates using the semiconservative method
  4. Identify key enzymes involved in DNA replication and their function
  5. Explain the DNA replication process
  6. Describe how telomerase work
  7. Describe the macrostructure of chromosomes

Remember that Mendel discovered a “heritable factor” that was responsible for diversity in nature. Unknown to Mendel, this heritable factor is the gene which is a section of DNA. Each human chromosome carries hundreds to thousands of genes, responsible for making various proteins. The model for the DNA was proposed by James Watson and Francis Crick in 1953.

Common features of the Watson and Crick model remain today:

  1. DNA is a double-stranded helix with the two strands connected by hydrogen bonds
  2. “A” bases are always paired with “T” bases and “G” bases are always paired with “C”
  3. The DNA double helix is antiparallel, i.e., the 5’ end is paired with the 3’ end of its complementary strand
  4. Nucleotides are linked together by their phosphate groups which binds the 3’ end of one sugar to the 5’ end of the next sugar
  5. The outer edges of the bases are exposed and available for hydrogen bonding. This allows the DNA to bind to other molecules including proteins that play a role in its replication and gene expression

Discovery of the Transforming Power of DNA

Evidence that DNA can transform bacteria came from work done by Fredrick Griffith in 1928. Griffith recognized that a pathogenic form of Streptococcus pneumonia killed mice when they were injected but the nonpathogenic form did not. However, if the pathogenic form was heat-treated and the cells mixed in with the nonpathogenic form, the nonpathogenic form became pathogenic. This was due to the transfer of a heritable substance, according to Griffith.

Figure 1. Summary of Griffith experiment. Image Source: Wiki Commons

Work by Oswald Avery led to the discovery in 1944 that DNA was the heritable substance. This was confirmed by studies done by Alfred Hershey and Martha Chase. In 1952, they performed an experiment to see if it is protein or DNA that enters the cell of bacteria and cause transformation. In this experiment, they made the protein in a phage (virus that infects bacteria) radioactive, and then mixed the phage with bacteria cells to cause the bacteria to become infected. They then separated the phage proteins from the bacteria by centrifugation. What they saw was that only the phage proteins were radioactive and the bacteria was not. So, clearly it was not protein being transferred to the bacteria that caused any heritable change.

In another part of the experiment, they only made the DNA in the phage radioactive and repeated the same steps. This time they recognized that only the bacteria was radioactive. Thus, the DNA was the heritable factor.   

Figure 2. Hershey and Chase experiment, 1952. Image source: Wiki commons

Additional evidence that the DNA is the genetic material came from Erwin Chargaff’s lab. According to “Chargaff rules”:

  1. Base composition varies among species (i.e. percentage of nucleotides across species)
  2. Within a specie the number of A and T nucleotides and C ang G nucleotides are equal. 

Structure of the DNA

After biologist confirmed that the DNA was the heritable factor, the challenge was to come up with a model that would accurately represent it and account for its functionality. The American, James Watson and Englishman, Francis Crick were the first to propose a model which they presented in the journal of Nature. The model was a double helix molecule with nitrogenous bases facing the interior of the helix and the two sugar-phosphate backbone running antiparallel to each other.  

Figure 3. Secondary structure of DNA. Image Source: Wiki Commons

Figure 4. The tertiary structure of DNA. The two chains are wrapped in the shape of a tertiary double helix structure with two groves, a minor and a major where proteins and drugs can interact with the functional groups on the bases that are exposed in the grooves. Image Source: Wiki Commons.

DNA Replication Model

Watson and Crick are well known not only for creating the correct model of the DNA but for also proposing how it replicates using the semiconservative method. The latter was a hypothesis which was proven by Matthew Meselson and Franklin Stahl in their 1958 publication titled “The replication of DNA in Escherichia coli” published in the Proceedings of the National Academy of Science.

There were three possible models that were being considered as the method by which DNA could replicate.

  1. Conservative Method: One DNA is a “photocopy” of the other i.e., you get an original and a full copy of the original
  2. Semiconservative Method: The DNA separates into two strands, and each strand is used as a template to copy another strand
  3. Dispersive Method: DNA gets cut into small pieces and then reattaches with new segments added in to create a patchwork of old and new DNA fragments
Figure 5. The three proposed DNA replication models

In their experiment, Meselson and Stahl designed a way to distinguish between the parent strand of the DNA and the daughter strand. They began by culturing the parent bacteria in a heavy nitrogen isotope (N15). This isotope was incorporated in the bacteria, causing it to have a heavy DNA. These parents were then removed from the heavy media and grown in a light media (N14). This caused the new daughter cells to incorporate the light nitrogen in their DNA. Therefore, their DNA was lighter. Since they had differences in density, the DNA from the parent DNA could be separated from the DNA of the daughter cells by centrifugation.

In the first replication, they observed a single layer of DNA. This is what would be expected if a semiconservative or the dispersive methods were at play. The conservative method would definitely not be the right method, since after the first replication, it would produce two layers (heavy and light) and not the one lay observed.

Now they needed to determine if the method was dispersive or semiconservative. Therefore, they did a second replication. If the method was dispersive, you would expect to see just one layer of DNA. However, if it was semi conservative, you should see two layers. This is exactly what they saw.   

Figure 6. The conservative replication method (first and second replication)

Summary of DNA Replication

We now have a good understanding of how DNA is replicated. The following is a summary of the mechanism. First, let us consider the key enzymes and proteins involved.

  1. Helicase – Enzyme that disrupts hydrogen bonds between bases, unwinds the helix, and separate the DNA into individual strands, thus creating a replication fork.
  2. Topoisomerase – Enzyme that breaks the DNA ahead of the replication fork, releases pressure and reconnects the DNA.
  3. RNA Primase – Enzyme that adds an RNA primer to one side of the DNA strands to allow copying to begin.
  4. DNA Polymerase – Enzyme that binds to the primer and creates a new DNA strand
  5. Ligase – Enzyme that joins DNA strands
  6. Exonuclease – Enzyme that removes RNA primer
  7. Single stranded binding (SSB) proteins – Proteins that bind the DNA strands during replication to prevent them from sticking back together after unwinding

Replication Steps

  1. Helicase unzips the double helix by breaking hydrogen bonds holding complementary bases.
  2. The unzipping of the DNA creates strain ahead of the replication fork. Therefore, topoisomerase breaks, untwists, and reconnects the DNA
  3. SSB proteins binds to separated strands to prevent them from sticking back together  
  4. A replication fork is created with the unzipping by helicase. One strand is oriented from 3’ to 5’ (leading strand) and the other from 5’ to 3’ (lagging strand)
  5. The Leading Stand
    1. A short sequence of RNA (RNA primer) binds to the leading strand with the help of primase. This is the starting point for DNA synthesis
    2.  DNA polymerase binds to the leading stands (3’to 5’) and adds new complementary nucleotides from the 5’ to the 3’ direction (read up, write down rule). This type of replication is called continuous. Note: DNA polymerase can only add DNA bases from the 5’ to 3’ direction
  6. The Lagging Strand
    1. The lagging strand runs in the opposite direction (5’to 3’) and so cannot be replicated continuously. Instead, replication is done in a series of small chunks called Okazaki fragments. First a primer is laid down and DNA polymerase adds new nucleotides from the 5’ to 3’ direction. As the helix opens, the strand at the 3’end (closer to the replication fork) is exposed. Therefore, the DNA polymerase will always have to be racing back up towards the replication fork to add more nucleotides to the growing strand. Thus, DNA replication on the lagging strand ‘lags’ behind and is discontinuous rather than continuous.
  7. Once both strands have been replicated, exonuclease remove primers from both strands
  8. DNA polymerase fills in the gaps where primer was removed
  9. DNA ligase seals Okazaki fragments 
  10. Two identical DNA molecules are made and the two DNA molecules wind to create double helix

Figure 7. DNA replication

Replicating the End of DNA Molecules

A small portion of the DNA cannot be replicated. This occurs at the tip of the chromosomes in a section known as the telomere. The telomere is a G-rich repeat section at the end of the DNA. In humans the actual sequence is TTAGGG. Why can’t this section be replicated? Well, remember that primers must be removed for DNA replication to be completed.

Figure 8. The shortening problem in somatic cells

Exonuclease is responsible for removing these primers. Once primers are removed. DNA polymerase fills in the gap from the 5’ to 3’ end. This leaves a gap at one of the 3’ end of the parent DNA. DNA polymerase cannot fill the gap since it needs to add nucleotides from the 3’ end, which is absent. This causes a shortening of the DNA every time it divides. This happens all the time in somatic cells, resulting in eventual failure to divide when the telomere becomes too short. Some cells have been able to overcome this challenge with the help of an enzyme called telomerase. These include cells in fetal tissue, adult germ cells (eggs and sperms), and tumor cells.

First, telomerase attaches to the overhang of the parental strand using its built-in RNA template, and adds nucleotides to elongate the parental strand.

Figure 9. Elongation of parent strand

After that, primase and DNA polymerase synthesize the complementary strand. 

Figure 10. Complementary nucleotides added to parent strand

In the end we will have the same problem that we started with. That is, we are left with a gap that cannot be filled due to the absence of a primer. However, this is not a problem. The overhanging sequence on the DNA is simply snipped off and removed. It is just junk DNA and therefore will not affect the cell’s function.

Figure 11. Extra (overhanding DNA removed.

Macrostructure of the DNA

Before we leave this chapter, let’s zoom out to get a bit of appreciation for the DNA as a macrostructure. As you know, the DNA is ultimately packaged in the form of a chromosome. But, what are other components of the chromosome apart from DNA? Imagine the DNA as a thread wrapped around spools. These “spools” are proteins called histones. Histones are rich with positively charged amino acids (arginine and lysine) which makes them bond tightly to the negatively charged DNA (Note: DNA is negative due to its phosphate groups). The ability for DNA to be compact tightly in a cell is due to histones. Histones are arranged in an octamer form. These appear like beads around which the DNA winds. There are five types of histone proteins – H1, H2A, H2B, H3, and H4. All but H1 proteins contribute to the formation of the octamer. H1 acts like a clip, fixing the DNA to the octamer. The combination of octamer and DNA thread around it, is called a nucleosome. This is the basic unit of DNA packing. The next level of packaging is the chromatosome, which include the combination of nucleosome, H1 protein and linker DNA (DNA connecting the nucleotides). Six H1 proteins associate to form a solenoid structure. The solenoid further stack on top of each other to form chromatin fiber. The chromatin fiber then undergoes supercoiling to form the highly dense chromatid structure in the chromosome.    

Textbook: Textbook: Reece, J. B., & Campbell, N. A. (2011). Campbell biology. Boston: Benjamin Cummings / Pearson.

Author

  • Courtney Simons

    Dr. Courtney Simons has served as a food science researcher and educator for over a decade. He holds a Bachelor of Science in Food Science and a Ph.D. in Cereal Science from North Dakota State University.