DNA: The Molecular Basis of Inheritance - Notes

Genetics Warm-Up Questions

• Draw and label a nucleotide.

• Why is DNA a double helix?

• What is the complementary DNA strand to: DNA: A T C C G T A T G A A C

Genetics Warm-Up Questions

• What was the contribution made to science by these people:

  • Hershey and Chase

  • Franklin

  • Watson and Crick

• Chargaff’s Rules: If cytosine makes up 22% of the nucleotides, then adenine would make up ___ % ?

• Explain the semiconservative model of DNA replication.

Genetics Warm-Up Questions

• What is the function of the following:

  • Helicase

  • DNA Ligase

  • DNA Polymerase (I and III)

  • Primase

  • Nuclease

• How does DNA solve the problem of slow replication on the lagging strand?

• Write the complementary DNA strand: 3’ T A G C T A A G C T A C 5‘

• What is the function of telomeres?

DNA: The Molecular Basis of Inheritance

• DNA nucleotide components:

  • Sugar (deoxyribose)

  • Phosphate group

  • Nitrogenous base (Adenine, Thymine, Guanine, Cytosine)

• Sugar-phosphate backbone

• 5’ and 3’ ends are indicated.

What You Must Know

• The structure of DNA.

• The major steps to replication.

• The difference between replication, transcription, and translation.

• The general differences between bacterial and eukaryotic chromosomes.

• How DNA is packaged into a chromosome.

Structure of DNA

• DNA = double helix

• “Backbone” = sugar + phosphate

• “Rungs” = nitrogenous bases

Structure of DNA Nitrogenous Bases

• Nitrogenous Bases:

  • Adenine (A)

  • Guanine (G)

  • Thymine (T)

  • Cytosine (C)

• Pairing:

  • purine + pyrimidine

  • A = T

  • G ≡ C

  • purine

  • pyrimidine

Structure of DNA

• Key features of DNA structure include:

  • Hydrogen bonds between base pairs of the two strands hold the molecule together.

  • Dimensions: 3.4 nm, 1 nm, 0.34 nm

Structure of DNA

• Antiparallel: one strand (5'→ 3'), other strand runs in opposite, upside-down direction (3' 5')

DNA Double Helix & Packaging

• DNA double helix is 2 nm in diameter.

• DNA is organized into nucleosomes (10 nm in diameter).

• Nucleosomes are “beads on a string” (10-nm fiber).

• Histones are proteins around which DNA is wrapped. Histone H1 is also present.

DNA Packaging

• 30-nm fiber

• Looped domains (300-nm fiber)

• Chromatid (700 nm)

• Replicated chromosome (1,400 nm)

• Loops connect to a scaffold.

• Metaphase chromosome

DNA Comparison

• Prokaryotic DNA:

  • Double-stranded

  • Circular

  • One chromosome

  • In cytoplasm

  • No histones

  • Supercoiled DNA

• Eukaryotic DNA:

  • Double-stranded

  • Linear

  • Usually 1+ chromosomes

  • In nucleus

  • DNA wrapped around histones (proteins)

  • Forms chromatin

Discovering Genetic Material

• Gregor Mendel (1822-1884):

  • Discovered the idea of heredity being more than simple blending of traits.

  • Studied traits passed down through generations of pea plants.

• Mendel’s conclusions:

  • Law of Segregation: ‘Factors’ of inheritance can be expressed in one of two ways (dominant or recessive) during the segregation of chromosomes during gamete production.

  • Law of Independent Assortment: Assortment of these various ‘factors’ occurred independently of one another during gamete formation.

• Chemical nature of heredity was still unknown.

• Scientists debated whether the heritable material was a protein or DNA.

Fredrick Miescher & Phoebus Levene

• Fredrick Miescher (1869):

  • First to isolate ‘Nuclein’ from white blood cells.

  • Determined genetic material had acid (nucleic acids) and alkaline (proteins) portions.

  • Called it ‘Nuclein’ because found only within the nucleus.

• Phoebus Levene (1909):

  • Studied structures of sugars and discovered ribose and deoxyribose.

  • Proposed two types of nucleic acids, each composed of long chains of nucleotides, distinguishable by their sugar.

  • Discovered DNA was made of long chains of repeating nucleotides each being composed of a sugar, phosphate and a nitrogenous base.

  • Incorrectly hypothesized that nucleic acids possessed equal amounts of each nucleotide along the chains.

• It was still unknown whether genetic material was made of protein or DNA.

The “Transforming Principle” - Frederick Griffith (1928)

• 1920's: Frederick Griffith trying to develop a vaccine against pneumonia.

• Took nuclear material from harmless live bacteria (pneumococcus) “rough” and mixed it with heat-killed pathogenic/virulent bacteria “smooth” pneumococcus. Then he injected the non-virulent version into mice. The mice died.

• This showed the process of transformation: the introduction of foreign DNA, by plasmid or virus, into a bacterial cell.

• Scientists suspect protein not the hereditary material.

• “Transforming Principle” - a substance passed from dead bacteria to live bacteria

The “Transforming Principle”

• Transformation = change in phenotype

• Something in heat-killed bacteria could still transmit disease-causing properties.

• Experiments:

  • Live pathogenic strain → mice die

  • Live non-pathogenic strain → mice live

  • Heat-killed pathogenic bacteria → mice live

  • Mix heat-killed pathogenic & non-pathogenic bacteria → mice die

Griffith's Experiment

• Hypothesis: Material in dead bacterial cells can genetically transform living bacterial cells.

• Experiment:

  • Kill virulent S strain bacteria by heating.

  • Mix dead S strain cells with living, nonvirulent R strain bacteria.

• Conclusion: A chemical substance from one cell is capable of genetically transforming another cell.

DNA is the “Transforming Principle” - Avery, McCarty & MacLeod (1944)

• Purified DNA & proteins from Streptococcus pneumonia bacteria

  • Treated the pathogenic bacteria with a protein destroying enzyme and placed it into a solution of non-pathogenic bacteria: transformation still occurred and mice died.

  • Treated pathogenic bacteria with a DNA destroying enzyme and placed it in a solution of non-pathogenic bacteria: transformation did not occur and mice lived.

  • Treated pathogenic bacteria with an RNA destroying enzyme and placed it in a solution of non-pathogenic bacteria: transformation still occurred and mice died.

• Conclusion: DNA is the inheritance factor and not protein!

Avery et al

• Purified S strain extracts to characterize the transforming principle.

• Material was resistant to proteases; it contained no lipid or carbohydrate.

• If DNA in the extract is destroyed, the transforming principle is lost.

• Pure DNA isolated from the S strain extract transforms R strain.

• Avery cautiously suggested that DNA was the genetic material.

• This was the first experimental evidence that DNA is the genetic material.

Avery, McCarty & MacLeod (1944)

• Conclusion: first experimental evidence that DNA was the genetic material

Avery, McCarty & MacLeod

• After Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty published the 1944 article, a number of their contemporaries immediately understood that transformation was the transfer of genetic material from one bacterium to another, and that the transforming substance, DNA, must be the genetic material.

• However, the team's somewhat tentatively stated conclusions were not met with complete acceptance.

• At the time, the belief that DNA was a monotonous chain of four repeating nucleotides-- structurally important but of little physiological interest--was still difficult to overcome.

• The belief that only proteins possessed the structural complexity necessary to carry hereditary information was pervasive among geneticists.

• Many of the scientists who had previously thought that genetic material was protein still believed that the effects of the transforming principle were perhaps due to some undetected protein associated with the DNA.

Confirmation of DNA - Hershey & Chase (1952)

• Classic “blender” experiment worked with radioactively labelled bacteriophage -viruses that infect bacteria

• Grew phages in 2 media, radioactively labeled with either

  • $^{35}S$ in the proteins of the virus

  • $^{32}P$ in the DNA of the virus

• Each type of phage was allowed to infect the bacteria. After which the infected bacteria samples were spun down to analyze for the presence and location of the radioactive isotopes.

• Findings: most of the radioactive phosphorous was found in the bacteria pellet and the radioactive Sulfur was found in the supernatant with the viruses.

• Conclusion: DNA and not protein enters the bacterial cells

• Why use Sulfur vs. Phosphorus?

Hershey & Chase Experiment

• Protein coat labeled with $^{35}S$

• DNA labeled with $^{32}P$

• Bacteriophages infect bacterial cells.

• Bacterial cells are agitated to remove viral protein coats.

• $^{35}S$ radioactivity found in the medium

• $^{32}P$ radioactivity found in the bacterial cells

• Radioactive marker found inside the cell: $^{32}P$

• Molecule carries viral genetic info: DNA

Hershey & Chase Experiment

• Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells.

• Agitate in a blender to separate phages outside the bacteria from the cells and their contents.

• Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube.

• Measure the radioactivity in the pellet and the liquid.

• The experiment showed that T2 proteins remain outside the host cell during infection, while T2 DNA enters the cell.

Hershey & Chase (1952)

Hershey and Chase (1952)

• Conclusion: DNA entered infected bacteria → DNA must be the genetic material!

Erwin Chargaff (1947)

• DNA composition: “Chargaff’s rules”

• DNA varies from species to species, but the DNA from the same species had the same nucleotide composition, but varied in sequence of bases.

• All 4 bases are not in equal quantity, but rather in definite proportions. bases present in characteristic ratio

• Humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8%

• Rules:

  • A = T

  • C = G

Edwin Chargaff (1947)

• Chargaff’s Rules:

  • DNA composition varies between species

• Ratios: %A = %T and %G = %C

Rosalind Franklin (1950’s)

• Worked with Maurice Wilkins

• X-ray crystallography = images of DNA

• Provided measurements on chemistry of DNA

Rosalind Franklin (1920-1958)

• Rosalind Franklin’s work with x-ray diffraction gave Watson and Crick the last clue to the structure of the double helix.

• She discovered that DNA was a double helix and that one complete turn of the helix occurred every 10 bases with a length of 3.4nm and that each nitrogenous base was 0.34nm apart.

• She found that the nitrogenous bases faced inwards and the sugar-phosphate back bone faced outwards.

• She was never recognized for her work until after her death. She died of cervical cancer.

Rosalind Franklin (1920-1958)

• A chemist by training, Franklin made original and essential contributions to the understanding of the structure of graphite and other carbon compounds before her appointment to King's College.

• James Watson's unflattering portrayal of Franklin in his account of the discovery of DNA's structure, entitled "The Double Helix," depicts Franklin as an underling of Maurice Wilkins, when in fact Wilkins and Franklin were peers in the Randall laboratory.

• Randall had given Franklin the task of elucidating DNA's structure through X-ray crystallography. With this technique, the locations of atoms in any crystal can be precisely mapped by looking at the image of the crystal under an X-ray beam.

• Franklin discovered (and was the first to state) that the sugar- phosphate backbone of DNA lies on the outside of the molecule and elucidated the basic helical structure.

• After Randall presented Franklin's data and her unpublished conclusions at a routine seminar, her work was provided - without Randall's knowledge - to her competitors at Cambridge University, Watson and Crick. The scientists used her data and that of other scientists to build their ultimately correct and detailed description of DNA's structure in 1953.

• Franklin was not bitter, but pleased, and set out to publish a corroborating report of the Watson-Crick model. Her career was eventually cut short by illness. Her role in the discovery wasn't recognized during her lifetime or after her death at age 37 due to cancer.

James Watson & Francis Crick (1953)

• Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules.

James Watson and Francis Crick

• Created a model of DNA connecting the phosphates to Carbon #5 and Carbon #3 hydroxyl groups forming the phosphodiester bonds holding the nucleotides along the chain together.

• Deduced that DNA was double stranded and that the strands ran opposite/antiparallel to each other (one in a 5’ to 3’ direction and the other in a 3’ to 5’ direction).

• Watson and Crick also determined that A=T and C=G and that this contributes to a uniform diameter (2nm) of the double helix.

• They found that Purines have a double ring structure and that Pyramidines have a single ring structure. Also that a purine always bonds with a pyramidine.

Other DNA research discoveries:

• Around the same time as Watson & Crick’s research, the structure of RNA was also being researched and found:

  • The sugar is ribose

  • Uracil is present and Thymine is absent

  • Single stranded and can fold back onto itself via complementation.

• Discoveries of nucleic acid structure helped to understand how genetic material is organized within cells.

• DNA cannot simply be left a mess inside the nucleus, but rather is organized, compact and protected from catalytic enzymes within the cell.

• An understanding between Prokaryotic and Eukaryotic genomes was developing helping to better understand these two types of living organisms.

DNA Replication

• Making DNA from existing DNA

• Three alternative models of DNA replication:

  • Conservative

  • Semiconservative

  • Dispersive

Meselson & Stahl Experiment

• Bacteria cultured in medium with $^{15}N$ (heavy isotope)

• Bacteria transferred to medium with $^{14}N$ (lighter isotope)

• DNA sample centrifuged after first replication and after second replication

Meselson & Stahl

• Different models of replication predict different results for the experiment described above.

  • Conservative

  • Semiconservative

  • Dispersive

Replication is semiconservative

• Parent molecule separates into strands.

• Each strand serves as a template for a new, complementary strand.

• Result: two identical daughter DNA molecules, each consisting of one parental strand and one new strand

Major Steps of Replication

• Helicase: unwinds DNA at origins of replication

• Initiation proteins separate 2 strands forming a replication bubble

• Primase: puts down RNA primer to start replication

• DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’ à 3’)

• Lagging strand grows in 3’à5’ direction by the addition of Okazaki fragments

• DNA polymerase I: replaces RNA primers with DNA

• DNA ligase: seals fragments together

DNA Replication Steps 1 & 2

1. Helicase unwinds DNA at origins of replication and creates replication forks

2. Initiation proteins separate 2 strands forming a replication bubble

DNA Replication Step 3

• Primase adds RNA primer

DNA Replication Step 4

• DNA polymerase III adds nucleotides in 5'-3' direction on leading strand

Replication on leading strand

• DNA polymerase III adds nucleotides in 5'-3' direction on leading strand

Leading strand vs. Lagging strand

• Overview of replication showing leading and lagging strands

Okazaki Fragments & DNA Ligase

• Okazaki Fragments: Short segments of DNA that grow 5’à3’ that are added onto the Lagging Strand

• DNA Ligase: seals together fragments

Bacterial DNA Replication Proteins and Their Functions

• Table outlines the functions of key proteins:

  • Helicase - Unwinds parental double helix at replication forks

  • Single-strand binding protein - Binds to and stabilizes single-stranded DNA until it can be used as a template

  • Topoisomerase - Relieves