DNA and Its Role in Heredity

DNA: The Genetic Material 1900-1910 – Embryologist & geneticists had associated traits (genes) with chromosomes EB Wilson & Nettie Stevens – sex & chromosome make-up TH Morgan – sex linked traits (genes)

DNA: The Genetic Material 1941 – George Beadle & Edward Tatum demonstrated that a single gene corresponded to a single enzyme

DNA: The Genetic Material 1920s - Frederick Griffith’s transformation experiment Virulent vs avirulent pneumococcal bacterial strains 1940-44 - Oswald Avery and colleagues identified the transforming substance of Griffith’s pneumococcal strain

Oswald Avery's Isolation of the Transforming Substance Figure: 10-03a Caption: Summary of Avery, MacLeod, and McCarty’s experiment, which demonstrated that DNA is the transforming principle.

DNA: The Genetic Material 1952 - Alfred Hershey & Martha Chase confirmed DNA is genetic material of viruses Bacteriophage - a virus infecting bacteria T2 a DNA phage of E. coli A DNA core packed in a protein coat

DNA: The Genetic Material Hershey-Chase experiment determined whether viral protein or DNA entered the bacterium & directed the synthesis of further viral particles

Figure 11.3 The Hershey–Chase Experiment

Questions Remaining About of DNA How does DNA cause the synthesis of specific proteins? How is DNA replicated between nuclear divisions? Structure of DNA ultimately provided insight to the answers

The Chemical Constituents of DNA 1859 – Friedrich Meidscher discovered and named nucleic acids (DNA) By 1940s known DNA was a polymer of nucleotides. DNA was assumed to be non-varying, repeating sequence of nucleotides unique to individual species 1950 - Erwin Chargaff carefully determined that individual percentages of A & T as well as G & C are equal and the A:T / G:C ratio varies among organisms

Nitrogenous Bases Figure: 10-07a Caption: (a) Chemical structures of the pyrimidines and purines that serve as the nitrogenous bases in RNA and DNA. (b) Chemical ring structures of ribose and 2-deoxyribose, which serve as the pentose sugars in RNA and DNA, respectively.

Deoxyribonucleic Acid Ribose Sugars Figure: 10-07b Caption: (a) Chemical structures of the pyrimidines and purines that serve as the nitrogenous bases in RNA and DNA. (b) Chemical ring structures of ribose and 2-deoxyribose, which serve as the pentose sugars in RNA and DNA, respectively. RNA Ribonucleic Acid DNA Deoxyribonucleic Acid

Figure: 10-08 Caption: Structures and names of the nucleosides and nucleotides of RNA and DNA.

Figure: 10-09 Caption: Basic structures of nucleoside diphosphates and triphosphates, as illustrated by thymidine diphosphate and adenosine triphosphate.

Figure: 10-10 Caption: (a) Linkage of two nucleotides by the formation of a C-3’–C-5’ (3’–5’) phosphodiester bond, producing a dinucleotide. (b) Shorthand notation for a polynucleotide chain.

DNA Structure Determination 1953 James Watson & Francis Crick Rosalind Franklin & R Gosling Maurice Wilkins Jerry Donohue Linus Pauling

Figure 11.4 X-Ray Crystallography Revealed the Basic Helical Structure of the DNA Molecule

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Figure 11.6 (b) DNA Is a Double Helix

Models of A & B DNA A-DNA B-DNA

Figure 11.7 Base Pairing in DNA Is Complementary

In 1953 DNA Structure Suggests Function Complementary Base Pairing Replication mechanisms Sequence of nucleotides Code corresponding to proteins in some way

Determining the DNA Replication Mechanism 1957 - Matthew Meselson and Franklin Stahl demonstrated DNA replication is semiconservative

Figure 11.9 The Meselson–Stahl Experiment

Theoretical Predictions Experimental Observation

Determining the DNA Replication Mechanism 1958 - Arthur Kornberg purified DNA polymerase & used it to replicate DNA in vitro Polymerization only worked with nicked, ds DNA templates Found that DNA polymerase requires a priming 3’- OH from which to initiate synthesis

The Mechanisms Overview of DNA Replication H-bonds between strands are broken, making each strand available to base pair with new nucleotide Sequence of new strand is directed by the sequence of the template strand – complementary base pairing Nucleotides are attached to the 3’ end of each growing strand

Figure 11.10 Each New DNA Strand Grows from its 5¢ End to its 3¢ End

DNA Synthesis is Bidirectional Two nascent, labeled strands at each fork means both parent strands serve as templates

Implication of Bidirectional Synthesis RULE: Polymerization can only happen in 5'3' direction Starting at 1 spot only 1 strand can serve as template but both strands do therefore, one strand synthesized continuously leading strand the other strand made discontinuously lagging stand

Okazaki Fragments If model is correct, should be able to find lagging strand fragments Discovered by Reiji & Tuneko Okazaki 1000-2000 nt long DNA fragments Begin with ~12 nucleotides of RNA Figure: 11-11 Caption: Opposite polarity of DNA synthesis along the two strands, necessary because the two strands of DNA run antiparallel to one another and DNA polymerase III synthesizes only in one direction (5’ to 3’). On the lagging strand, synthesis must be discontinuous, resulting in the production of Okazaki fragments. On the leading strand, synthesis is continuous. RNA primers initiate synthesis on both strands. Priming

Figure 11.15 Many Proteins Collaborate at the Replication Fork

Mechanics of DNA Synthesis Figure: 11-13 Caption: Summary of DNA synthesis at a single replication fork. Various enzymes and proteins essential to the process are shown.

The Molecular Mechanisms of DNA Replication Enzymology DNA polymerase Primase Helicase Topisomerase replication complex recognizes an origin of replication on a chromosome.

The Molecular Mechanisms of DNA Replication Replication occurs from many origins simultaneously Large chromosomes can have hundreds of origins of replication The region replicated from a single origin is called a replicon The complex of enzymes is the replisome

Figure 11.17 The Lagging Strand Story (Part 2)

Figure 11.18 Telomeres and Telomerase

DNA Proofreading and Repair Proofreading by DNA polymerase minimizes errors Mutation rate of most eukaryotic DNA polymerases ~ 10-8 1 error every 1x108 bp Mutation rate in prokaryotic cells is higher ~10-6 – 10-7 DNA damage UV, free radicals, etc.. Repair mechanisms frequently excise damaged sequences and resynthesize DNA to repair damage

Practical Applications of DNA Replication The technique of DNA sequencing hinges on the use of modified nucleosides called dideoxynucleotides (ddNTPs). ddNTPs lack both 2’ and 3¢ hydroxyl groups 3’ OH is site of addition of next nucleotide Like dNTPs, ddNTPs are picked up by DNA polymerase and added to a growing DNA chain But once added, chain elongation is terminated

Figure 11.21 Sequencing DNA

DNA Sequencing Primer annealed

DNA Sequencing Primer extended

Practical Applications of DNA Replication The polymerase chain reaction (PCR) technique for making multiple copies of a DNA sequence. PCR cycles through three steps: Double-stranded fragments of DNA are heated to denature them into single strands. A short primer is annealed DNA polymerase catalyzes the production of new DNA strands

Figure 11.20 The Polymerase Chain Reaction Cycle 1 Cycle 2 Cycle 3