Lecture 2 Structure of DNA

Discoveries Hereditary material exists – Mendel’s plant genetics Chemical nature of DNA Physical nature of DNA Structure of DNA

Discoveries: The Chemical Nature of DNA 1869—Fredrich Miescher named the chemical nuclei contained nuclein. Other chemists discovered it was acidic and named it nucleic acid. It was soon realized that there were two types of nucleic acids: DNA and RNA. Early in the 20 th century, 4 types of nucleotides were discovered.

Physical Nature of DNA – A Physical Unit You can see it – experiments demonstrated that inheritance is on chromosomes You can move it from one place to another – experiments demonstrated that DNA can carry new traits into an organism You can change it with other physical processes – traits can be changed by physical processes

Once Morgan showed that genes are located on chromosomes, proteins and DNA were the candidates for the genetic material. Until the 1940s, the specificity of function of proteins seemed to indicate that they were the genetic material. However, this was not consistent with experiments with microorganisms, like bacteria and viruses. The Search for Genetic Material Leads to DNA Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

I. Bacterial Transformation is Mediated by DNA Experiment by Frederick Griffith – 1928 – Demonstrated first evidence that genes are molecules – Two different strains of Streptococcus pneumoniae Non-pathogenic = Avirulent = ROUGH cells (R) Pathogenic = virulent = SMOOTH (S) – Smooth outer covering = capsule – Capsule = slimy, polysaccharide – Encapsulated strains escape phagocytosis

Continued. – The capsule alone did not cause pneumonia Heat-killed S strain was avirulent Ability to escape immune detection and multiply – When heat-killed S strain was mixed with living R strain  the mouse dies of pneumoniae Encapsulated strain (S) recovedred from dead mouse  Now a live strain The R strain had somehow acquired the ability to produce the polysaccharide capsule – Transformation – Ability to produce coat was an inherited trait  Daughter cells also produced capsule

The Experiment

First Demonstration of Transformation – Uptake of genetic material from an external source resulting in the acquisition of new traits (phenotype is changed) – Griffith’s expriment was the earliest document evidence of transformation

What was this transforming agent? Avery, MacLeod and McCarty defined the transforming agent of Griffith’s experiment as DNA (1944) – Chemical components of heat-killed S strain bacteria were purified and co-injected with live R strain Polysaccharide/Carbohydrate Lipids Protein Nucleic acids – DNA – RNA

Oswald Avery, Maclyn McCarty and Colin MacLeod announced that they found that only DNA transformed the cells. To replicate, a virus infects a host cell and takes over the cell’s metabolic machinery. Viruses that specifically attack bacteria are called bacteriophages or just phages. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings

II. Viral DNA is Transferred into Cells During Infection – The Hershey-Chase Experiment (1952) T2 Bacteriophage studies – Bacteriophage = viruses that infect bacteria – Major chemical components = DNA and protein – Escherichia coli infected with T2 produce thousands of new viruses in the host cell Host cell lyses and phage are released

Hershey Chase Experiment Determination of whether DNA or protein was directing synthesis of new phage particles – Viral proteins were radioactively labeled with: 35 S by growing T2-infected bacteria in 35 S-methionine = 1 st Batch – Amino acid labeling – DNA does not contain any sulfur atoms 32 P by growing T2-infected bacteria in 32 -P – Nucleic acid labeling – Amino acids do not contain phosphorous

Continued.. – Radioactively labeled viruses were isolated from the culture and used to REINFECT new host cells Batch 1 = protein labeled Batch 2 = DNA labeled – Blender used to disrupt phage on surface of bacteria from cells and their cytoplasmic components  then centrifuged Supernatant?? (Protein never entered the cell) Pellet?? (DNA injected into the cell)

III. Chargaff’s Rules Erwin Chargaff (1947) provides more evidence that DNA = genetic material – Analysis of base composition of DNA compared between different organisms Nitrogenous bases – Adenine (A) – Thymine (T) – Guanine (G) – Cytosine (C) – Conclusions of Chargaff DNA composition is species specific The amounts of A,G,C and T are not the same between species – Ratios of nitrogenous bases vary between species

– This diversity strengthened argument that DNA is the molecular basis of inheritance – Chargaff’s Rules Amount of A = T Amount of G = C

IV. X-Ray Crystallography Data Provides James Watson and Francis Crick with Insight into DNA Structure The Race is On – Linus Pauling – Maurice Wilkins and Rosalind Franklin – Watson and Crick X-ray Crystallography defined – Diffracted X-rays as they pass through a crystallized substance – Patterns of spots are translated by mathematical equations to define 3-D shape

Watson and Crick Discovered the Structure of DNA by Building Models of X-ray Data Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA. – X-rays are diffracted as they passed through purified, crystallized DNA. – The diffraction pattern can be used to deduce the three- dimensional shape of molecules. James Watson learned from their research that DNA was helical in shape.

X-ray Chrystallograph of DNA The diffraction pattern obtained by Franklin and Wilkins showed a X pattern which hinted of a 2 stranded helical form

The helical turn of DNA correlates to the horizontal lines in the picture which measures to 34 Angstroms. They also calculated that the gap between based pairs was 3.4 A as measured on the distance from the center of the X to the ends. Simple math deduced that there are 10 nucleotides per turn.

Franklin and Wilkins also showed that the sugar phosphate backbones were found to be on the outside of the helix. The hydrated and dry forms of DNA showed that water could easily come in and bind to DNA, a fact that could only happen if the feature showed sugar phosphate backbones being on the outside.

Rosalind Franklin’s data provide clues about DNA’s 3-D shape – Helix – Width = 2 nm  probably two strands (DOUBLE HELIX) – Nitrogenous bases = 0.34 nM apart – One turn every 3.4 nM (10 base pairs per turn)

The arrangement of the three major components in nucleic acid polymers was already well known – but the 3-D shape was still unclear – Sugar phosphate backbone – Bases

Putting the hydrophobic nitrogenous bases on the inside, and the sugar-phosphate groups on the outside was a stable arrangement

Base pairing was worked out by trial and error – The distance between the sugar-phosphate backbone groups is constant Therefore purine-purine or pyrimidine- pyrimidine were not allowed because spacing would be in inconsistent with data – Purines = A and G (two organic rings) – Pyrimidines – C and T ( one organic ring)

Purine-pyrimidine base pairing would be consistent with X-ray data

Hydrogen bonding between purines and pyrimidines established the appropriate pairs and reinforced Chargaff’s Rules – 2 hydrogen bonds between A and T – 3 hydrogen bonds between G and C

Nature 171: – April 1953 Watson JD and Crick FC (1953) Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid – Nobel Prize awarded to three men – Watson, Crick and Wilkins

Slides from The Structure and Function of DNA - Part I Transformation, Transfection, Chargaff’s Rules and the Double Helix by Sherry Fuller-Espie, 2002