DNA

Chapter 15: DNA

Genetic Material Definition

  • To be considered ‘genetic material’, it must:

    • Replicate itself: This means that genetic material can make copies of itself during cell division and inheritance.

    • Direct and control living processes: It must have the ability to control and regulate biological activities essential for life.

Model of Genetic Inheritance

  • Mendelian Genetics: Developed in the early 1900s, focusing on the inheritance patterns of traits.

    • Inheritance of Genes: One copy of each gene is inherited from each parent, resulting in zygotes containing two alleles for each trait.

    • Chromosomes: The structures that carry genes, which are composed of DNA and proteins.

    • Meiosis: The process of cell division where the distribution of chromosomes during meiosis in gamete formation explains Mendel's laws of inheritance.

Chromosome Composition

  • Chromosomes are made of:

    • Proteins

    • DNA

The Question of Genetic Material

  • There was a significant debate over whether proteins or DNA served as genetic material. Early thoughts favored proteins because:

    • Complexity of Proteins: Proteins are chemically diverse with many varieties possible.

    • Despite evidence of a transforming substance affecting heredity, the exact nature of that substance was unknown at the time.

Frederick Griffith's Experiment

  • Objective: Develop a vaccine against Streptococcus pneumoniae.

    • Types of Strains:

    • S strain: Smooth appearance due to a capsule, virulent (capable of causing disease).

    • R strain: Rough appearance, nonvirulent (does not cause disease).

    • Experiment Summary:

    • Nonvirulent (R strain) does not cause sickness; Virulent (S strain) kills mice.

    • Mice injected with heat-killed S strain survive.

    • Mice injected with R strain mixed with heat-killed S strain die, indicating something was transferred from R to S strain.

  • Conclusion: A transforming element was transferred that caused the R strain to become virulent.

Avery, MacLeod, and McCarty Experiments

  • This work built on Griffith's findings to identify the transforming element.

  • Method:

    • Isolated and purified components from the virulent S strain after heat-killing it. Tested various enzymes:

    • Proteases: Destroy proteins; transformation still occurs.

    • RNases: Destroy RNA; transformation still occurs.

    • DNAses: Destroy DNA; transformation fails, indicating DNA is the transforming substance.

Hershey-Chase Experiment

  • Conducted to provide further evidence that DNA is the genetic material.

  • Method:

    • Used bacteriophages (viruses that infect bacteria).

    • Phages were labeled using:

    • Radioactive sulfur: Labels proteins.

    • Radioactive phosphorus: Labels DNA.

  • Observation: Cells glowed green (phosphorus, indicating DNA), not yellow (sulfur, indicating proteins).

  • Conclusion: Confirmed that DNA is indeed the genetic material of all living things.

DNA Structure

  • Recall Monomers/Polymers:

    • DNA is a polymer composed of (deoxyribo)nucleotide monomers.

  • Structure of a Nucleotide:

    • Phosphate Group

    • 5 Carbon Deoxyribose Sugar

    • Nitrogenous Bases: Adenine (A), Cytosine (C), Thymine (T), Guanine (G)

Components of DNA Backbone

  • Nitrogenous Bases: Two categories exist.

    • Purines: Adenine (A) and Guanine (G) - Structures: Two-ringed.

    • Pyrimidines: Cytosine (C) and Thymine (T) - Structures: One-ringed.

  • Phosphodiester Linkages:

    • The connection between nucleotides; links the 3' end of one sugar to the 5' end of another via phosphodiester bonds.

Base Pairing Rules

  • Base Pairing:

    • Adenine (A) pairs with Thymine (T) using 2 hydrogen bonds.

    • Guanine (G) pairs with Cytosine (C) using 3 hydrogen bonds.

    • In RNA, Adenine pairs with Uracil (U) instead of Thymine.

  • Importance of Base Pairing Rules:

    • Ensures complementary strands of DNA are formed.

    • Maintains accuracy during transcription processes.

Chargaff’s Rule

  • Observation by Erwin Chargaff: The amount of adenine (A) equals thymine (T) and cytosine (C) equals guanine (G) in all organisms tested.

  • Implications:

    • This implies: A + G = T + C

    • Thus, the percentage of purines equals the percentage of pyrimidines.

Chargaff’s Rule Application (Mathematical Example)

  • Given a DNA molecule with 180 base pairs and 20% adenine:

    • % adenine = 20%; % thymine = 20%

    • Total % of A and T = 40%; remaining % = 60%.

    • Distributed equally among guanine and cytosine, thus:

    • % guanine = 30% and % cytosine = 30%.

    • Total bases = 180 imes 2 = 360.

    • Thus, cytosine nucleotides = 0.30 imes 360 = 108.

Rosalind Franklin's Contributions

  • Conducted X-ray diffraction studies demonstrating that DNA is a helical structure.

  • Proved DNA's helical nature through X-ray diffraction patterns generated by her samples.

Watson and Crick Model

  • Published the accepted model of the structure known as the DNA double helix.

  • Key Features of their Model:

    • DNA exists as a double helix with antiparallel strands.

    • Strands held together by hydrogen bonds between bases (base pairs).

    • Adenine pairs with Thymine through 2 hydrogen bonds; Guanine pairs with Cytosine through 3 hydrogen bonds.

    • The strands are complementary, meaning each side must have the appropriate sequence opposite it.

Temperature and DNA Stability

  • Consider two separate DNA strands:

    • Strand A: has a Guanine/Cytosine (G/C) content of 60%.

    • Strand B: has a G/C content of 30%.

  • Question: Which strand will require a higher temperature to denature?

    • Answer: Strand A will require a higher temperature due to its higher G/C content, which forms three hydrogen bonds compared to the two bonds in A/T pairs, making it more stable.

3.Semiconservative nature of DNA

  • Each DNA strand can serve as a template for making a complementary strand 

  • Semiconservative = 1 old strand + 1 new strand 

Meselson and Stahl 

  • Used E.coli as their model organism to determine semiconservative nature of DNA 

  • 1. Grew E.coli in a medium with 15N.

  • 2. Transferred E.Coli into a new medium with 14N.

  • After a while, they found that the medium had equal amounts of 15N and 14N. Meaning DNA replicates semiconservatively. 

4. DNA replication 

Summary 

  • Requires the coordinated activity of many enzymes and other proteins 

  • Begins at the origin of replication site, which creates a replication bubble

    • Usually only 1 origin of replication in circular, prokaryotic DNA

    • Eukaryotic cells usually have several

We will focus primarily on eukaryotic DNA replication

  • Both strands are replicated at the same time on both sides of the replication bubble, which produces a Y-shaped replication fork on either side. 

  • The replication fork moves in as synthesis proceeds. 

Step #1 Unwind the DNA

  • Enzyme: DNA helicase ‘unzips’ the DNA 

  • Other enzymes involved: 

    • ssDNA (single-stranded DNA) binding proteins hold the DNA open

    • Topoisomerases break and rejoin strands, resolving knots and strains that may occur.

Step # 2 Adding nucleotides 

  • Problem? 

  • Recall: DNA is antiparallel 

  • The enzyme that adds nucleotides (to make a new strand) is called DNA polymerase (DNAP)

  • DNA polymerase knows where to bind because of DNA primase 

  • DNAP is picky. This enzyme only moves (synthesizes DNA) in 5’ to 3’ direction. It reads the strand in the 3’ to 5’ direction.

  • Therefore, DNAP adds nucleotides to the 3’ end of DNA only!!!

  • Introducing: Leading strand, lagging strand, and okazaki fragments 

  • The replication fork opens more as replication continues

  • DNAP adds to the leading strand continuously 

  • DNAP adds to the lagging strand discontinuously generating Okazaki fragments 

Step#3 Sealing the DNA back together 

  • Enzyme: DNA ligase

  • Seals the Okazaki fragments together 

  • Also joins together DNA strands at the end of DNA replication 

Is DNA replication always perfect?

  • No. 

  • To prevent errors, DNAP proofreads the DNA to make sure it lays down the right nucleotide 

  • Initial error rate 1:100,000

  • Final error rate 1:100,000,000

  • If an error goes unnoticed initially DNA repair can occur 

  • Cells have repair mechanisms to fix most mistakes that get through

Telomeres?

  • The physical ends of chromosomes

  • Present another problem for DNA replication 

  • Part of the DNA at the end of a eukaryotic chromosome goes uncopied in each round of replication, leaving a single-stranded overhang.

  • Over multiple rounds of cell division, the chromosomes will get shorter and shorter as this process repeats

  • Telomerases - enzymes that can generate longer telomeres

  • Higher activity levels observed in cancer cell (so they live longer) 

V. DNA packaging in chromosomes 

How long is DNA

  • 20,000 genes in the human genome 

  • DNA is 6 feet long in one of your cells 

  • Do this in all of your cells in you body- that number becomes 67 billion miles long 

  • We need to be able to efficiently package DNA within chromosomes

Nucleosomes

  • Main packaging mechanism for eukaryotic DNA 

  • Made of 8 protein subunits, acts like a “spool” for the DNA “thread” 

  • Gives an appearance of “beads” on a “string” 

  • Proteins = positively charges histones 

Histone H1 and scaffolding proteins 

  • Nucleosomes pack into a 30nm chromatin fiber 

  • The 30nm fibers then form looped domains that are 300nm wide 

  • Relevance: This maximizes the amount of DNA we can pack into our cells