DEOXYRIBONUCLEIC ACID (DNA)

OBJECTIVE: to examine the basic structure and properties of DNA CONTENT 1. Definition of DNA 2. Primary structure of DNA 3. Double helix structure of DNA 4. Super helix structure of DNA 5. Hybridization The Discovery of the Molecular Structure of DNA The Double Helix A Scientific Breakthrough The sentence "This structure has novel features which are of considerable biological interest" may be one of science's most famous understatements. It appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix, the molecule that carries genetic information from one generation to the other. Nine years later, in 1962, they shared the Nobel Prize in Physiology or Medicine with Maurice Wilkins, for solving one of the most important of all biological riddles. Half a century later, important new implications of this contribution to science are still coming to light. I. DEFINITION OF DNA

Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms. The main role of DNA molecules is the long-term storage of information and instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information. II. A. STRUCTURE OF DNA DNA is a polymer of deoxyribonucleoside monophosphates; 1. Each nucleotide is made up of a sugar, one or more phosphate groups and a base. 2. the bases are adenine, guanine, cytosine, and thymine; 3. the bases are on the inside and sugar-phosphate backbone on outside; 4. base pairs are formed through hydrogen bonding; 5. the nucleotides are linked through phosphodiester bonds(sugar-acid) 6. the DNA molecule is a right handed helix; 6. bases are perpendicular to the helical axis stacked on top of each other(superpos ou paral) and interacting through hydrophobic interactions and van der Waals interactions;

8. approximately 10 base pairs per(double) helical turn; 9. every DNA molecule has a specific sequence of nucleotides called its primary structure where genetic information is stored. B. Most DNA in a cell exists as the Watson-Crick double helix which is known as B form DNA.

Figure 1: Francis Crick and David Watson (1953)

Figure 2: Bruce Wilkins

In 1951, the then 23-year old biologist James Watson travelled from the United States to work with Francis Crick, an English physicist at the University of Cambridge. Crick was already using the process of X-ray crystallography to study the structure of protein molecules. Together, Watson and Crick used X-ray crystallography data, produced by Rosalind Franklin and Maurice Wilkins at King's College in London, to decipher DNA's structure.

Figure 3: The original DNA model by Watson and Crick Major features of DNA The two strands of the double helix are anti-parallel, which means that they run in opposite directions. The sugar-phosphate backbone is on t he outside of the helix, and the bases are on the inside (Figure 4 ) Two base pairs can be formed - either an adenine-thymine pair that form a twohydrogen bond together, or a cytosine-guanine pair that form a three-hydrogen bond. The base pairing is thus restricted. This restriction is essential when the DNA is being copied: the DNA-helix is first denatured in two long stretches of sugar-phosphate backbone with a line of free bases sticking up from it. Each half will then be the template for a new, complementary s trand.

The coding regions in the DNA strand, the genes, make up only a fraction of the total amount of DNA. The stretches that flank the coding regions are called introns, and consist of non-coding DNA. Today, biologists and geneticists believe that this non-coding DNA may be essential in order to expose the coding regions and to regulate how the genes are expressed.

Figure 5: The sugar-phosphate backbone is on the outside and the four different bases are on the inside of the DNA molecule.

C. DNA supercoiling 1. a supercoil is when the double-helix twists around itself; 2. supercoils can be positive or negative but natural DNAs exist in the negative supercoiled form; 3. DNA can be supercoiled if it is circular or if it is linear and has fixed ends; 4. supercoiled DNA is more compact than relaxed DNA; 5. negatively supercoiled DNA molecules are easier to unwind than relaxed molecules; 6. DNA unwinding is required for replication and transcription. D. Topoisomerases are enzymes that catalyze changes in DNA supercoiling 1. Type I topoisomerases function by breaking a phosphodiester bond of one strand, passing the other strand through the break and resealing the break--they can only remove supercoils; 2. Type II topoisomerases function by breaking both strands and passing a double strand region through the break before resealing the break. This process requires ATP; 3. topoisomerases are targets of numerous chemotherapeutic drugs: adriamycin, VP16 (tenoposide), VM26 (etoposide), camptothecin. E. Nucleases--enzymes that catalyze hydrolysis of phosphodiester bonds in nucleic acids 1. exonucleases cleave terminal nucleotides from either the 5' or 3' end of a polynucleotide; 2. endonucleases cleave in the interior of nucleic acid molecule.

3. restriction enzymes are endonucleases that cleave at specific sequences of DNA. F. DENATURATION AND RENATURATION OF DNA

1. Denaturation is the conversion of the double stranded form of DNA into single stranded form a. DNA can be denatured by heat or alkaline treatment; b. The temperature at which half the DNA is unwound is defined as the melting temperature (Tm); --Tm is dependent on the GC content of the DNA, on the solvent, and on the ionic strength. 2. Renaturation--under proper conditions, complementary single-stranded nucleic acids can renature into a double-stranded form. 3. Denaturation and Renaturation is the basis of hybridization experiments--this type of analysis is central to recombinant DNA technology and gene manipulation. 4. Hybridization

Hybridization is the process, discovered by Alexander Rich, of combining complementary, single-stranded nucleic acids into a single molecule. Nucleotides will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. This is called annealing. However, due to the different molecular geometries of the nucleotides, a single inconsistency between the two strands will make binding between them more energetically unfavorable. Measuring the effects of base incompatibility by quantifying the rate at which two strands anneal can provide information as to the similarity in base sequence between the two strands being annealed. Annealing may be reversed by heating the double stranded molecule of DNA (or RNA or DNA:RNA) to break the hydrogen bonds between bases and separate the two strands. This is called melting or denaturation.