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The Structure and Function of DNA - Part I 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 – 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 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 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 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 Determination of whether DNA or protein was directing synthesis of new phage particles – Viral proteins were radioactively labeled with: 35S by growing T2-infected bacteria in 35Smethionine = 1st Batch – Amino acid labeling – DNA does not contain any sulfur atoms 32P by growing T2-infected bacteria in 32-P – Nucleic acid labeling – Amino acids do not contain phosphorous – 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 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 sugarphosphate groups on the outside was a stable arrangement Base pairing was worked out by trial and error – The distance between the sugarphosphate backbone groups is constant Therefore purine-purine or pyrimidinepyrimidine 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: 737-738 – April 1953 Watson JD and Crick FC (1953) Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid. 1962 – Nobel Prize awarded to three men – Watson, Crick and Wilkins The Structure and Function of DNA - Part II DNA Replication: Utilization of Numerous Enzymes, Proteins and RNA Primers I. The Molecular Mechanism of DNA Replication The copying process of DNA is related to nitrogenous base pairing rules – Parent DNA molecule consists of two strands Complementary – A pairs with T; G pairs with C The two strands run in antiparallel directions (DNA has polarity) – The two DNA strands separate and serve as templates to direct the synthesis of “new” complementary strands – New nucleotides are inserted along the template – A pairs with T; G pairs with C – Each nucleotide that is added is covalently attached to the previous one Enzyme = DNA Polymerase Sugar-phosphate backbone of new strand is formed The linear DNA sequence exists in many states – Each gene has its own UNIQUE sequence – Knowing the sequence of one strand, you can deduce the sequence of the other (complementarity) II. Three Models of DNA Replication Conservative model – Parent molecule remains the same – Completely new copy of the double helix is made Semiconservative model – Parent strands separate and serve as templates for new strand synthesis – Hybrid molecules are made Dispersive model – New strands contain a mixture of old molecules and newly synthesized molecules Messelson and Stahl Experiment Supports the Semi-Conservative Model of DNA Replication III. DNA Replication Involves a Complex Assembly of Proteins and Enzymes Human haploid genome = 3 x 109 bp – ~>1000 X more complex than Escherichia coli – The Human Genome Project has sequenced the entire genome of our species Worldwide effort – International Collaborations – The DNA synthesis phase (interphase) during mitosis lasts only a few hours despite its huge size – Replication of the DNA sequence is very accurate Mutation rate ~1/109 errors The Sequence of Events 1. Beginning of replication occurs at the origins of replication – Prokaryotic cells (e.g. E. coli) contain only one origin – Eukaryotic cells contain thousands of orgins on each chromosome – Proteins bind to origins and pry open the two strands – A replication bubble appears at the site of strand separation and new DNA synthesis Replication forks forma at the ends of the bubbles – Replication occurs in both directions on the two strands But ALWAYS in the 5’ 3’ direction per strand – Replication bubbles fuse 2. DNA polymerases add on new nucleotides to the growing DNA strand – One nucleotide is added at a time to the 3’-OH group of the previous nucleotide – The 3’-OH group of the ribose sugar is covalently linked to the nucleoside triphosphate forming a phosphodiester bond – Two phosphate groups are liberated – energy is released (PPi – pyrophosphate) 3. How to resolve the replication fork dilemma of antiparallel strands – DNA strands have polarity (antiparallel) – DNA polymerase can only add new nucleotides to the 3’ end of the terminal deoxyribose – Synthesis always progresses in the 5’ 3’ direction – Leading strand is synthesized continuously DNA polymerase progresses as DNA is unzipped One continuous polymer is made – Lagging strand is synthesized discontinuously Synthesized in opposite direction Synthesized AWAY from the replication fork Initiated as a series of short segments called Okazaki fragments (100-200 bases long) Okazaki fragments are joined together by DNA ligase (covalent phosphodiester bonds between fragments) 4. RNA primers are required for initiation of DNA synthesis – DNA polymerase can add nucleotides only to 3’OH group of an already existing nucleotide paired to its complement on the other strand – Q: How do things get started? – A: RNA primers are made by an enzyme called PRIMASE ~10 nucleotides long primers H-bonds to template and provides substitute for DNA polymerase Leading strand requires only one RNA primer Lagging strand requires one RNA primer for every Okazaki fragment – RNA primers are removed by specific enzymes and replaced with DNA nucleotides Gaps are sealed with DNA ligase 5. Helicases and single-stranded binding proteins are important components of DNA synthesis – Helicases unwind the double helix and separate the two templates Smoothing of twists Breaking of H-bonds – Single-stranded binding proteins stabilize the DNA for replication The Telomere Problem Eukaryotic cells have a problem replicating the 5’ ends of daughter DNA strands – The ends of chromosomes contain 100-1000 repeating (TTAGGG)n segments – Result: Ends of DNA get shorter and shorter in most dividing somatic cells Older people have shorter telomeres Some cells have a solution: – Telomerase Enzyme + RNA fragment – catalyzes the extension of the ends RNA = template for new telomere pieces – Examples: Germ cells Some cancerous cells Immortalized cultured cells The Structure and Function of DNA – Part III The Central Dogma I. Breaking the Genetic Code – Finding the Central Dogma An “RNA Club” organized by George Gamow (1954) assembled to determine the role of RNA in protein synthesis Radioactive tagging experiments demonstrate intermediate between DNA and protein = RNA Vernon Ingram’s research on sickle cell anemia (1956) tied together inheritable diseases with protein structure – Link made between amino acids and DNA – RNA movement tracked from nucleus to cytoplasm site of protein synthesis DNA Transcription RNA Protein Translation Genetic code determined for all 20 amino acids by Marshal Nirenberg and Heinrich Matthaei and Gobind Khorana – Nobel Prize – 1968 3 base sequence = codon