L8_2025_-_DNA

-Page 3: Lecture Overview

• Semester 2 Dates: January 23, 27, 28, 2025

• Lecture Topics:

  • Lecture 8: DNA as a genetic information store

  • Lecture 9: RNA and gene expression

  • Lecture 10: Protein synthesis

Page 4: Learning Objectives for Lecture 8 (DNA)

• Understand evidence that DNA carries genetic information.

• Describe the anti-parallel structure of DNA and the importance of base-pairing.

• Explain the Meselson-Stahl experiment demonstrating semi-conservative DNA replication.

• Detail how proteins like DNA polymerase facilitate DNA replication.

• Discuss the accuracy of DNA replication and the implications of mutations.

Page 5: Historical Context of Protein Phosphorylation

• Key Figures:

  • Philip Cohen (2002) on protein phosphorylation.

  • Crebs and Fischer (1992) on protein kinase A (PKA).

  • Carl and Gerty Cori (1947) on glycogen metabolism.

  • Earl Sutherland (1971) on cAMP signaling.

  • Gilman and Rodbel (1994) on G proteins.

• Scientific inspiration by Lefkowitz/Kobilka (2012) on G protein-coupled receptors.

Page 6: Key Experiments Contributing to DNA Understanding

• Mendel (1860s): Genetic traits in peas established the concept of inheritance.

• Miescher (1860s): Isolated nuclein from white blood cells – leading to the discovery of DNA.

• Morgan (1900s): Confirmed that genes are located on chromosomes via Drosophila studies.

Page 7: Transformation Concept

• Definition: Transformation is a change in genotype and phenotype due to assimilation of external DNA by a cell.

• At the time of Griffith's work, the inheritance factor's identity remained unknown, mainly attributed to proteins.

Page 8: DNA carries genetic information

• Oswald T. Avery: Conducted experiments that systematically destroyed lipids, carbohydrate, proteins and ribonucleic acid of virulent bacteria, but transformation still occurred.

• used deoxyribonuclease to destroy DNA and transformation was blocked.

• Summary

  • genetic info is carried in DNA, eve with lipids, carbs, proteins and ribonucleic acid is destroyed, transformation still occurred

Page 9: Confirmatory Evidence for DNA

• Chemical evidence confirmed DNA's role as genetic material was supported by viral studies indicating only DNA entered bacterial cells, not proteins (Hershey-Chase experiment).

Page 10: Composition of DNA

• Erwin Chargaff (1947): Determined that DNA composition varied between species. (molecular diversity)

• within a species the DNA from all cells had the same composition

• G=C, A=T

Page 11: Structure of Nucleotides

• DNA Composition: A polymer of nucleotides consisting of a sugar (deoxyribose), a phosphate, and nitrogenous bases (A, T, G, C).

  • A and G are purines (2 fused rings)

  • C and T are pyrimidines (1 ring)

  • the sugar is 5-carbon sugar 2 ‘ - deoxyribose

Page 12: Discovery of DNA Structure

• Rosalind Franklin (1952): X-ray diffraction revealed DNA's helical nature and base spacing.

• this allowed Watson to deduce width of helix and spacing of nitrogenous bases along it

Page 13: Watson and Crick’s Model

• Base Pairing: Watson and Crick established specific base pairing rules (A with T, G with C) due to hydrogen bonds, leading to the proposal of double helix structure.

  

Page 14: Features of the Watson and Crick Model

• Key Structural Features:

  • Sugar-phosphate backbone.

  • Anti-parallel strands (5’-3’ and 3’-5’).

  • 10 base pairs per helical turn.

  • Bases are non polar hydrophobic on the inside and the polar hydrophilic phosphate is on the outside

  • space filling models show a major and minor groove in the double helix

Page 15: DNA Replication Mechanism

• Overview: The Watson-Crick model implies strands serve as templates for new strand synthesis via base-pairing.

• The two strands need to unwind and separate to expose the bases to allow the synthesis of new DNA to occur.

Page 16: Meselson and Stahl

• Concept: DNA replication is semi-conservative; as opposed to ‘conservative’ or ‘dispersive‘ models

• verified by Meselson and Stahl through isotopic labelling studies.

• Conservative model

  • the two parental strands re-associate after acting as templates for new strands, restoring the parental double helix

  • original double helix is intact and unchanged

• Semiconservative Model

  • the two strands of the parental molecule separate, each functions as a template for synthesis of a new complementary strand.

  • consists of one parental and one newly synthesised strand

• Dispersive Model

  • each strand of both daughter molecules contains a mixture of old and newly synthesised DNA

  • parental DNA is mot kept intact but dispersed into both strands

Page 17: Experimental Methodology in Replication Studies

• Key Features of the Experiment:

  • Used E. coli as a model organism which could grow on N and C

  • nitrogen isotopes used for differentiating old and new DNA. N¹⁵ N¹⁴

  • Density gradient centrifugation to separate the two types N¹⁵ is 1% denser

  • the results were only consistent with the semi-conservative model

Page 18: Initiation of Replication

• DNA replication begins at origins specific to prokaryotes and eukaryotes

• proteins recognise the DNA sequence and bind to it, opening up the double helix, in the replication bubble, there are Y-shaped replication forks where the new strands are being elongated

  • replication starts at multiple sites where the parental strands separate to form replication bubbles

  • the bubbles expand laterally as DNA replication proceeds in both directions

  • the replication bubbles fuse and synthesis of the daughter strands are complete

Page 19: Role of DNA Polymerases

• Function: DNA polymerases elongate strands by incorporating nucleotides from triphosphate sources (ATP, GTP etc…)

• Mechanics of Elongation: A two phosphate unit (pyrophosphate) is split out as the chain is extended by each nucleotide.

  • nucleotides can ONLY be added to the free 3’ end and never the 5’ end

Page 20: Directionality of DNA Replication

• Due to anti-parallel strands and enzymes only being able to extend chains in only one direction (5’→3’), the two trands cannot both be replicated continuously

• resulting in a leading strand (continuous replication) and a lagging strand (discontinued replication)

Page 21: Lagging Strand Formation

• Okazaki Fragments

  • typically 100-200 nucleotides in length and are later joined by DNA ligase

• DNA polymerase can only extend a pre existing strand, the cell makes a short primer of RNA about 10 nucleotides long, this is then replaced by DNA.

• DNA polymerase adds nucleotides to the 3' end of this primer, elongating the new DNA strand. This results in Okazaki fragments.

Page 22: Key Proteins in DNA Replication

• Functions of Key Proteins:

  • Helicase: Unwinds DNA at replication forks.

  • Single-strand binding proteins: Stabilise unwound DNA.

  • Topoisomerase: corrects over-winding, swivelling and rejoins

  • Primase: Synthesises RNA primers at 5’ end of leading strand, synthesises 5’ end of okazaki fragment on lagging strand

  • DNA polymerases: Elongate leading and lagging strands.

  • DNA ligase: Joins 3’ end of DNA fragments on leading strand and joins okazaki fragments on lagging strand

Page 23: Lecture 8 Summary

• Review discoveries by Mendel, Miescher, Morgan, Griffith, Avery, Hershey-Chase, Chargaff, Meselson-Stahl, Watson-Crick.

• Understand DNA polymerase functions on leading and lagging strands.

• Memorise names and roles of proteins in replication.