Chapter 9 Notes: DNA and RNA Structure, DNA Replication, and Chromosome Structure

9.1 Properties and Identification of the Genetic Material

• Four criteria for genetic material:

  • Information: Must contain information to construct an entire organism.
  • Replication: Must be accurately copied.
  • Transmission: Must be passed from parent to offspring and from cell to cell during cell division.
  • Variation: Account for differences between individuals and species.

• Griffith’s Bacterial Transformation Experiments:

  • Frederick Griffith worked with Streptococcus pneumoniae bacteria.
  • Smooth (S) strains: secrete capsules, typically deadly.
  • Rough (R) strains: do not secrete capsules, typically survivable.
  • Mix of live type R and heat-killed type S bacteria injected into mice resulted in death, and living type S bacteria were isolated.
  • Postulation: A substance (genetic material) from dead type S cells transformed type R cells into type S.

• Avery, MacLeod, and McCarty:

  • Used Griffith’s observations to identify DNA as the genetic material.
  • Purified DNA could convert R to S.
  • Enzymes that break down DNA, RNA, or protein were used to degrade potential contaminants.

9.2 Nucleic Acid Structure

• DNA and RNA are nucleic acids, polymers of nucleotides that are responsible for the storage, expression, and transmission of genetic information.

• Levels of complexity:

  • Nucleotides: the building blocks.
  • Strand: a linear polymer.
  • Double helix: two strands twisted together.
  • Chromosomes: DNA associated with different proteins.
  • Genome: the complete complement of genetic material in an organism.

• Nucleotides:

  • Pentose sugar, a phosphate group, and a nitrogen-containing base.
  • Base attached to the 1ʹ carbon atom, and a phosphate group attached to the 5ʹ carbon.

• Strand:

  • Covalent bonds, called phosphodiester bonds, link nucleotides together.
  • Sugar-phosphate backbone formed by sugar in one nucleotide linked to a phosphate group in the next nucleotide.
  • Bases project away from the backbone.
  • Directionality based on the orientation of the sugar molecules.
  • The 5ʹ end has a free phosphate group and the 3ʹ end has a free hydroxyl group.

• DNA Double Helix:

  • Double-stranded helix with outer backbone and bases on the inside.
  • Stabilized by H-bonds between base pairs.
  • Base pairing is specific (AT/CG rule).
  • Strands are complementary to each other.
  • Strands are antiparallel.
  • One complete turn is 3.4 nm and comprises ~10 base pairs.
  • Contains major groove and minor groove; the major groove provides a binding site for many proteins.

9.3 Discovery of the Double-Helix Structure of DNA

• X-ray Diffraction Studies:

  • Rosalind Franklin analyzed DNA diffraction patterns which indicated:
    • a helical structure
    • a uniform diameter (~2nm)
    • diameter too big to be a single strand

• Base Composition Analysis:

  • Erwin Chargaff analyzed the base composition of DNA from different species.
  • The amount of adenine was similar to the amount of thymine, and the amount of cytosine was similar to guanine.

• Watson and Crick:

  • Synthesized work of others to discover the structure of DNA.
  • Used x-ray diffraction data of Franklin, base ratio data of Chargaff, and the ball-and-stick modeling approach of Linus Pauling.
  • Published their work in 1953.

9.4 Overview of DNA Replication

• Meselson and Stahl:

  • Considered 3 proposed mechanisms of DNA replication:
    • Semiconservative mechanism.
    • Conservative mechanism.
    • Dispersive mechanism.
  • Original strands are parent strands, and newly made strands are daughter strands.
  • Experiment used isotope labeling to differentiate among the three proposed DNA replication mechanisms.
    • E. coli grew in an environment with ^{15}N to label DNA, then switched to an environment with ^{14}N.
    • Samples collected after each generation were consistent with the semiconservative mechanism.

• DNA Replication:

  • The 2 parental strands are separated and serve as template strands for synthesizing daughter strands.
  • The result is two double helices with the same base sequence as the original DNA.

9.5 Molecular Mechanism of DNA Replication

• Origin of Replication:

  • A site within a chromosome serves as a starting point for DNA replication.
  • DNA strands are unwound, forming a replication bubble.
  • Two replication forks are formed within this bubble.
  • Replication proceeds outward from the replication forks in both directions (bidirectional replication).
  • Bacterial chromosomes are relatively small and circular with a single origin of replication.
  • Eukaryotic chromosomes are larger and have a linear structure with multiple origins of replication.

• Proteins Required:

  • DNA helicase: uses energy from ATP to break H-bonds between base pairs.
  • DNA topoisomerase and single-strand binding proteins are responsible for fork formation and movement.
  • DNA polymerase: covalently links nucleotides together.
    • Incoming nucleotides are triphosphates; pyrophosphate is broken away, and the energy released is coupled to the formation of a new bond between nucleotides.
    • Cannot begin synthesis on a bare template strand; it can only extend a pre-existing strand.
    • Synthesizes DNA in a 5’ to 3’ direction.
  • DNA primase: makes a complimentary primer of RNA (10 to 12 nucleotides in length) that can be extended by DNA polymerase.

• Leading and Lagging Strands:

  • The leading strand is made continuously, extending in the same direction that the replication fork is moving.
  • The lagging strand is made as a series of small Okazaki fragments, extending in the opposite direction as the replication fork.

• DNA Replication Accuracy:

  • Permanent mistakes in DNA replication are extraordinarily rare (ex: 1 mistake per 100 million nucleotides in bacteria).
  • High fidelity due to:
    • Hydrogen bonds between A/T and C/G are more stable than between mismatched pairs.
    • DNA polymerase is unlikely to catalyze bonds between nucleotides if a mismatched base pair occurs.
    • DNA polymerase can proofread to remove mismatched pairs.

9.6 Molecular Structure of Eukaryotic Chromosomes

• Chromosome Compaction:

  • Eukaryotic chromosomes must be folded and compacted to fit inside the nucleus.
  • Chromosome: a discrete unit of genetic material.
  • Chromatin: a complex of DNA and proteins that makes up eukaryotic chromosomes.

• Nucleosomes:

  • Eukaryotic DNA is first compacted by wrapping around histone proteins, which forms structures called nucleosomes.
  • The nucleosome is a repeating structural unit that is 11 nm in diameter.
  • Histone proteins contain many positively charged amino acids that interact with the negatively charged phosphates of DNA.
  • Linker regions of DNA connect adjacent nucleosomes.

• 30-nm Fiber:

  • Nucleosomes are organized into a more compact structure that is 30 nm in diameter.
  • Histone H1 and other proteins are important in the formation of the 30-nm fiber.

• Loop Domains:

  • A third level of compaction involves interactions between the 30-nm fibers and proteins to form loop domains.
  • Proteins called CTCF can form loops.
    • CTCF = CCCTC binding factor, these proteins bind to a larger DNA sequence that contains CCCTC
  • SMC proteins can also form loops when the SMC proteins form a dimer.
    • SMC stands for structural maintenance of chromosomes
  • CTCF and SMC proteins may also be preset together at loop domains

• Heterochromatin and Euchromatin:

  • Heterochromatin: highly compacted.
  • Euchromatin: less condensed.
  • In heterochromatin, genes are usually inactive.
  • In nondividing cells, each chromosome occupies its own discrete region in the nucleus and usually does not overlap with the territories of other chromosomes.

• Maximum Compaction:

  • During cell division, each chromosome becomes entirely condensed.
  • All euchromatin is compacted to heterochromatin, which is further condensed to form the final (maximally compacted) metaphase chromosome.