3. DNA unzipping

Why are we here? What's life
all about? Is God really real, or
is there some doubt? [...] Or are
we just simply spiraling coils of
self-replicating DNA?

Monty Python's
The Meaning of Life (1983)

The discovery of DNA and its relation with the transmission of the genetic information was a process that took almost a century. The research involved many scientists with different backgrounds. All this prolonged investigation led to one of the most beautiful scientific achievements of the 20th century: the central dogma of molecular biology. Formulated by Francis Crick in 1958 [20], it eventually gave a physico-chemical basis to Mendelian genetics.

In 1865, Gregor Mendel set the foundations of genetics. He postulated the existence of genes as the basic units of heredity. So he established the rules of the heredity without knowing its physico-chemical basis. In 1869, Friedrich Miescher reported the existence of a new substance (nowadays known as nucleic acids) in the nucleus of the cells. Latter in 1882 Walther Flemming discovered the mitosis (i.e., the eukaryotic cell division) and the chromosomes. However, it was not until 1902 when two scientists (Theodor Boveri and Walter Sutton) independently identified the chromosomes as the carriers of the genetic information. This idea was experimentally verified by Thomas Morgan in 1910. Latter, the investigations focused on the composition of the chromosomes. In 1919, Phoebus Levene discovered that the nucleic acids were composed of nucleotides. However, he thought that the nucleotides were too simple to carry all the genetic information. At that time, most biologists believed that the proteins of the chromosomes were the carriers of the heredity. In 1944, the Avery-MacLeod-McCarty experiment -which was a revised version of Griffith experiment (1928)- concluded that DNA was the actual carrier of genes. The experiment consisted in mixing living avirulent bacteria with a large inoculum of lethal heat-killed cells and injecting it into mice. The experimentalists understood that the DNA was the substance that induced the transformation of the avirulent bacteria into the lethal ones, which produced the death of the mice. In the early 1950's, Erwin Chargaff discovered that the amount of guanine in DNA is equal to cytosine and the amount of adenine is equal to thymine. He established the two so-called Chargaff's rules that helped to predict the base-pairing and the structure of DNA. In 1953, Watson and Crick [1] proposed the double helix structure of DNA, using a X-ray image taken by Rosalind Franklin. All this period of investigations concluded in 1968, when Khorana, Holley and Nirenberg were awarded with the Nobel prize for their discovery of the genetic code.

Since then, molecular biology has experienced a tremendous development. The processes of the cell have been explored in great detail and our knowledge about them is nowadays enormous. Nevertheless, the central dogma of molecular biology is still valid. It summarizes the very essential molecular mechanisms related to the flow of the genetic information.

Although some RNA (such as ribozymes) perform important cellular functions, generally speaking the proteins are the biomolecules that determine the structure and the function of each cell: different cells have different proteins. All proteins are made from a sequence of 20 different elementary components called aminoacids. So each cell is capable of producing its proteins by gathering and bonding the correct sequence of aminoacids. The sequence of aminoacids to produce one protein is coded in the DNA. Now, the DNA is a biomolecule composed of a sequence of 4 types of nucleotides. Each group of 3 nucleotides codes for one amino acid and this is the ultimate physical support of the genetic information. Figure 3.1 shows how the genetic information is transferred from the DNA to RNA in order to build the proteins.

Figure 3.1: Central dogma of molecular biology [89]. The DNA stores the genetic information. Replication is the process by which this information is duplicated during the cell division. Transcription consists in the synthesis of a sequence of RNA from DNA. Finally, translation is the process by which proteins are synthesized according to the sequence of the RNA transcript. Translation is then carried out by ribosomes.

DNA forms a double helix that must be split for the cellular machinery to access the genetic information coded in the bases. Cells have specific molecular complexes to carry out such tasks. For instance DNA polymerases and their associated proteins are in charge of DNA replication. Similarly, RNA polymerases perform the transcription of DNA into RNA. During the last decades of the 20th century, scientists were able to reproduce these processes in vitro. Besides, the development of single-molecule techniques allowed them to directly manipulate and study these processes.

This chapter focuses on the process of DNA unzipping by force. DNA unzipping essentially consists in pulling apart the two strands of DNA by exerting mechanical forces on the extremities of the molecule. After describing the structure of DNA, this chapter focuses on the unzipping experiments and the models introduced to understand them.

JM Huguet 2014-02-12