Chapter 14: The Molecular Basis of Inheritance and DNA Replication

Important Experiments Leading to the Structure of Genetic Material

Early 20th-century scientists sought to identify the genetic material responsible for inheritance. Key experiments established DNA as the hereditary molecule.

• Fredrick Griffith’s Experiment (Streptococcus pneumoniae):

  • S strain: Smooth, virulent, kills mice.

  • R strain: Rough, non-virulent, mice survive.

  • Heat-killed S strain: Non-virulent, mice survive.

  • Mix of heat-killed S strain and live R strain: Mice die; live S strain recovered. This demonstrated transformation—the uptake of genetic material by a cell, changing its phenotype.

• Hershey and Chase Experiment (Bacteriophage T2):

  • Bacteriophage T2: Virus infects bacteria; composed of DNA and protein.

  • Radioisotopes: P32 labels DNA (phosphate groups), S35 labels protein (sulfur in amino acids).

  • Experiment: Phages grown with either isotope, allowed to infect bacteria. Only P32 entered cells, proving DNA is genetic material.

• Erwin Chargaff’s Experiment and Chargaff’s Rules:

  • Experiment: Measured nucleotide composition in DNA from various species.

  • Data Pattern: Amount of adenine equals thymine; guanine equals cytosine.

  • Chargaff’s Rules:

    1. DNA composition varies between species.

    2. A = T and G = C in DNA.

• Wilkins and Franklin’s X-ray Diffraction Studies:

  • Rosalind Franklin: X-ray diffraction revealed DNA’s helical structure, width, and spacing of bases.

• Watson and Crick’s DNA Model:

  • Structure: Double helix, two antiparallel strands (5' to 3' and 3' to 5').

  • Monomeric subunits: Nucleotides (sugar, phosphate, nitrogenous base).

  • Bonds: Phosphodiester bonds link nucleotides; hydrogen bonds between complementary bases (A-T, G-C).

  • Arrangement: Strands run in opposite directions; base pairing follows Chargaff’s Rules.

DNA Replication

Before cell division, DNA is copied to ensure genetic continuity. The process is highly regulated and accurate.

• Semi-conservative Model: Each new DNA molecule contains one old strand and one new strand.

  • Major Steps: Unwinding, priming, synthesis, and joining.

  • Outcome: Two identical DNA molecules, each with one parental and one newly synthesized strand.

• Chromosome Differences:

  • Prokaryotes: Single, circular chromosome; small size.

  • Eukaryotes: Multiple, linear chromosomes; larger size.

• Start Sites for DNA Replication:

  • Origin of replication: Specific DNA sequence where replication begins.

  • Structure: Replication bubble forms; two replication forks move outward.

  • Prokaryotes: Single origin per chromosome.

  • Eukaryotes: Multiple origins per chromosome.

• Proteins and Structures at the Replication Fork:

  • Topoisomerase: Relieves supercoiling ahead of fork.

  • Helicase: Unwinds DNA helix.

  • Primase: Synthesizes RNA primer.

  • Primer: Short RNA sequence for DNA polymerase attachment.

  • Single-stranded binding proteins: Stabilize unwound DNA.

  • DNA polymerases: Synthesize new DNA strands.

• Substrates and Reaction:

  • Substrates: Deoxyribonucleoside triphosphates (dNTPs).

  • Reaction: Addition of dNTPs to growing strand, catalyzed by DNA polymerase.

  • Equation:

• Replication of Antiparallel Strands:

  • Leading strand: Synthesized continuously toward replication fork.

  • Lagging strand: Synthesized discontinuously away from fork in Okazaki fragments.

  • Similarities: Both require primers and DNA polymerase.

  • DNA Polymerase I: Removes RNA primers, replaces with DNA.

  • DNA Ligase: Joins Okazaki fragments.

• DNA Polymerase I vs III:

  • DNA Polymerase III: Main enzyme for DNA synthesis.

  • DNA Polymerase I: Removes primers, fills gaps.

• Accuracy Mechanisms:

  • Proofreading: DNA polymerase checks and corrects errors during synthesis.

  • Mismatch repair: Enzymes correct errors after replication.

Nucleotide Excision Repair

Cells repair DNA damage before replication to maintain genetic integrity. Nucleotide excision repair fixes specific mutations.

• Type of Mutation: Fixes thymidine dimers and bulky lesions caused by UV light.

• Steps and Enzymes:

  1. Damage detected.

  2. Excision of damaged DNA by endonucleases.

  3. DNA polymerase fills gap.

  4. DNA ligase seals nick.

• Disease: Xeroderma pigmentosum—defective repair leads to extreme sensitivity to UV and increased cancer risk.

Telomeres and Telomerase

Linear chromosomes in eukaryotes have telomeres at their ends, which protect genetic information during replication.

• Telomere Features: Repetitive DNA sequences at chromosome ends; prevent loss of coding DNA.

• Replication: Telomeres shorten with each cell division.

• Telomerase: Enzyme that extends telomeres; active in germ cells, stem cells, and cancer cells.

Packaging DNA into the Nucleus

DNA is tightly packed in the nucleus to fit and regulate gene expression. Chromatin structure is key to this process.

• Chromosome Composition: DNA and protein.

• Chromatin: Complex of DNA and proteins (mainly histones).

• Types of Chromatin:

  • Euchromatin: Less condensed, transcriptionally active.

  • Heterochromatin: Highly condensed, transcriptionally inactive.

• Levels of DNA Packing:

  1. DNA double helix

  2. Nucleosome (DNA wrapped around histone octamer)

  3. Chromatin fiber (30 nm)

  4. Looped domains

  5. Chromosome (most condensed)

• Histones and Nucleosomes: Histones are proteins that DNA wraps around to form nucleosomes, the basic unit of chromatin structure.

Example: In human cells, telomerase is active in embryonic stem cells, allowing indefinite cell division, but inactive in most somatic cells, leading to aging.

Additional info: DNA repair mechanisms are essential for preventing mutations that can lead to diseases such as cancer. The semi-conservative model was confirmed by the Meselson-Stahl experiment (not listed but relevant).