Molecular Basis of Inheritance

DNA: The Substance of Inheritance

• Hereditary Information and Development: DNA serves as the primary substance of inheritance. The hereditary information encoded within DNA is responsible for directing the development of several organismal traits:

  • Biochemical Traits: The chemical processes and molecular functions within an organism.

  • Anatomical Traits: The physical structure and organization of the body.

  • Physiological Traits: The functional aspects of organs and biological systems.

  • Behavioral Traits: The patterns of action and response to the environment.

• Replication and Cellular Presence: Hereditary information is precisely reproduced in all cells of the body during the biological process of DNA replication.

The Scientific History of Genetic Material Discovery

• Early 20th Century Foundations: In the early 1900s, T.H. Morgan’s research group demonstrated that genes are located on chromosomes. This discovery identified the two components of chromosomes—DNA and protein—as the primary candidates for genetic material.

• The Case for Proteins (Pre-1940s): Until the 1940s, scientists generally believed proteins were the genetic material because:

  • Proteins were recognized as a class of macromolecules possessing high heterogeneity and specificity of function, which were seen as essential requirements for hereditary material.

  • Nucleic acids were poorly understood; their physical and chemical properties appeared too uniform to account for the vast multitude of specific inherited traits found in every organism.

• Frederick Griffith (1928): The understanding of DNA's role in heredity began with Griffith's research on two strains of Streptococcus pneumoniae:

  • Strains: One pathogenic (disease-causing) strain and one harmless strain.

  • Transformation: When Griffith mixed the heat-killed remains of the pathogenic strain with living cells of the harmless strain, some of the living cells became pathogenic.

  • Definition of Transformation: A change in genotype and phenotype due to the assimilation of foreign DNA.

• Oswald Avery (and colleagues): Later identified the "transforming substance" observed by Griffith as DNA. Despite this, skepticism remained in the biological community due to the prevailing belief in protein complexity.

• Hershey and Chase (1952): Provided definitive evidence that DNA is the genetic material using the T2 bacteriophage (a virus that infects bacteria).

  • The Experiment: They demonstrated that during infection, only the DNA of the T2 phage enters the E. coli cell, while the protein coat remains outside.

  • Conclusion: The injected DNA of the phage provides the necessary genetic information for reproduction.

• Erwin Chargaff (1950): Reported that DNA composition varies between different species, adding credibility to DNA as the genetic material.

  • Chargaff’s Rules:

    1. Base composition of DNA varies between species.

    2. In any given species, the percentages of Adenine ($A$) and Thymine ($T$) bases are equal, and the percentages of Guanine ($G$) and Cytosine ($C$) bases are equal.

The Structural Model of DNA

• The Double Helix Discovery: In 1953, James Watson and Francis Crick determined the structure of DNA.

• Methodological Contributors:

  • Maurice Wilkins and Rosalind Franklin: Used X-ray crystallography to study molecular structure.

  • Rosalind Franklin: Produced a critical X-ray crystallographic image of DNA that allowed Watson to deduce that DNA was helical.

• Watson and Crick’s Deductions:

  • Determined the width of the helix and the spacing of the nitrogenous bases based on Franklin's images.

  • Determined that the molecule is composed of two strands forming a double helix.

  • Antiparallel Backbones: Watson built a model where the sugar-phosphate backbones run in opposite directions ($5'$ to $3'$ and $3'$ to $5'$).

• Physical Dimensions of DNA:

  • Diameter: $2\,\text{nm}$.

  • Base Spacing: Bases are $0.34\,\text{nm}$ apart.

  • Helical Turn: One full turn occurs every 10 base pairs, spanning $3.4\,\text{nm}$.

• Nucleotide Structure: DNA is a polymer (polynucleotide) made of nucleotide monomers. Each nucleotide consists of:

  • A nitrogenous base (Adenine, Guanine, Thymine, or Cytosine) with a hydrocarbon ring structure.

  • A five-carbon ($5\text{-C}$) pentose sugar called deoxyribose.

  • A phosphate group.

• Base Pairing Principles:

  • Watson and Crick initially thought bases paired like-with-like ($A$-$A$), but this did not create a uniform width.

  • They discovered that pairing a Purine (double-ring) with a Pyrimidine (single-ring) resulted in the uniform width consistent with X-ray data.

  • Specific Pairing: $A$ pairs only with $T$ (forming 2 hydrogen bonds), and $G$ pairs only with $C$ (forming 3 hydrogen bonds). This explains Chargaff’s rules ($A=T$ and $G=C$).

DNA Replication: The Basic Concept

• Complementary Strands: Because the two strands are complementary, each serves as a template for reconstructing the other.

• Semiconservative Model: Proposed by Watson and Crick, stating that when a double helix replicates, each of the two daughter molecules will have one old strand (derived from the parent) and one newly synthesized strand.

• Alternative (Incorrect) Models:

  • Conservative Model: The two parental strands rejoin.

  • Dispersive Model: Each strand is a segmented mixture of old and new DNA.

The Mechanism of DNA Replication

• Origins of Replication: Replication begins at specific sites called origins, where the two DNA strands are separated, forming a replication "bubble."

  • Replication Fork: A Y-shaped region at each end of the bubble where parental strands are unwound.

  • Eukaryotic Replication: Long DNA molecules in eukaryotes have multiple replication bubbles that eventually fuse, speeding up the overall process.

• Essential Proteins in Unwinding:

  • Helicases: Enzymes that untwist the double helix at replication forks.

  • Single-strand binding proteins (SSBPs): Bind to and stabilize single-stranded DNA to prevent re-annealing.

  • Topoisomerase: Relieves strain caused by tight twisting ahead of the replication fork by breaking, swiveling, and rejoining DNA strands.

• Synthesizing New DNA:

  • Primer Requirements: DNA polymerases cannot initiate synthesis; they can only add nucleotides to an existing chain. Therefore, synthesis begins with a short RNA primer (5–10 nucleotides long).

  • Primase: An enzyme that starts an RNA chain from a single RNA nucleotide, using the parental DNA as a template.

• DNA Polymerases:

  • Catalyze the elongation of new DNA at the replication fork.

  • Add nucleotides to the $3'$ end of a preexisting chain.

  • Elongation Rate: Approximately $500\,\text{nucleotides/sec}$ in bacteria and $50\,\text{nucleotides/sec}$ in human cells.

• Energy for Synthesis: Each added nucleotide is a nucleoside triphosphate (like dATP).

  • dATP vs. ATP: dATP contains deoxyribose; ATP contains ribose.

  • Chemistry: As each monomer joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate ($PP_i$).

Antiparallel Elongation and Strand Differences

• Directionality: A new DNA strand can only elongate in the $5'$ to $3'$ direction because DNA polymerase adds nucleotides only to the free $3'$ end.

• Leading Strand: Synthesized continuously, moving toward the replication fork. Requires only one primer.

• Lagging Strand: Synthesized discontinuously, moving away from the replication fork.

  • Okazaki Fragments: The series of segments that make up the lagging strand.

  • Fragment Length: $100\text{--}200$ nucleotides in eukaryotes; $1,000\text{--}2,000$ nucleotides in E. coli.

• Completion of the Lagging Strand:

  • DNA Polymerase I: Removes RNA primers and replaces them with DNA nucleotides.

  • DNA Ligase: Joins the remaining gaps between DNA fragments by forming covalent bonds.

• DNA Replication Machine: A complex of proteins that may be stationary, "reeling in" parental DNA and "extruding" newly made daughter molecules.

Proofreading, Repair, and Evolution

• Accuracy: Initial errors are rare, and the final error rate in a completed DNA molecule is only 1 in 10 billion.

• Mechanisms of Repair:

  • Proofreading: DNA polymerases check newly made DNA and replace incorrect nucleotides.

  • Mismatch Repair: Other enzymes correct errors in base pairing that were missed during proofreading.

  • Nucleotide Excision Repair: A nuclease cuts out damaged stretches of DNA (caused by X-rays or chemicals). DNA polymerase then fills in the gaps using the undamaged strand as a template, and DNA ligase seals the ends.

• Hereditary Defects: A defect in repair enzymes is associated with forms of colon cancer, as errors accumulate faster.

• Evolutionary Significance: Mutations (permanent sequence changes) are the source of genetic variation upon which natural selection operates. While mostly rare after repair, they allow for biological diversity.

Replicating the Ends of DNA (Telomeres)

• The End-Replication Problem: For linear DNA, the replication machinery cannot complete the $5'$ ends of daughter strands, leading to shortening with each round of replication.

• Telomeres: Special repetitive, non-coding nucleotide sequences at the ends of eukaryotic chromosomes.

  • Function: They do not contain genes; they postpone the shortening of essential genetic information. Shortening is linked to the aging process.

• Telomerase: An enzyme that catalyzes the lengthening of telomeres in germ cells to ensure gametes have full-length chromosomes.

  • Clinical Relevance: Not active in most somatic cells, but often inappropriately active in cancer cells, making it a potential target for cancer therapy.

Chromosome Structure and Packaging

• Bacterial Chromosomes: Double-stranded, circular DNA molecules associated with a small amount of protein. DNA is "supercoiled" in a region called the nucleoid.

• Eukaryotic Chromosomes: Linear DNA molecules associated with a large number of proteins.

• Chromatin: A complex of DNA and protein found in the nucleus of eukaryotic cells.

• Levels of Packing:

  • Histones: The first level of protein packing.

  • Nucleosomes: Consist of DNA wound twice around a protein core of 8 histones (two of each main histone type).

• Interphase Organization:

  • Heterochromatin: Highly condensed chromatin (e.g., at centromeres). Its dense packing makes the genetic information inaccessible for transcription.

  • Euchromatin: More dispersed, less compacted chromatin that is accessible for transcription.

  • Dynamic Nature: Chromosomes are modified, condensed, or loosened as needed for various cellular processes.

Genetic Engineering and DNA Technology

• Nucleic Acid Hybridization: Use of complementary base pairing of one strand of nucleic acid to another. This forms the foundation of genetic engineering (direct manipulation of genes).

• DNA Cloning: Generating multiple identical copies of a specific DNA segment.

  • Plasmids: Small circular DNA in bacteria that replicate independently.

  • Recombinant DNA: A plasmid containing DNA inserted from another source.

  • Cloning Vector: A plasmid used to carry a foreign gene into a host cell for cloning.

• Restriction Enzymes: Bacterial enzymes that cut DNA at specific restriction sites.

  • Restriction Fragments: The pieces of DNA resulting from the cuts.

  • Sticky Ends: Staggered cuts that produce single-stranded ends which can bond with complementary sticky ends of other fragments.

  • Gel Electrophoresis: A technique used to separate nucleic acid fragments based on their length.

• Polymerase Chain Reaction (PCR): Amplifies a specific target DNA segment exponentially.

  • Taq Polymerase: A heat-stable DNA polymerase used in PCR.

  • Three Steps: Heating (denaturation of strands), Cooling (annealing of primers), and Extension (extension of primers in the $5'$ to $3'$ direction).

  • Applications: PCR is used to provide fragments for cloning or to amplify DNA from ancient sources like the Woolly Mammoth.

• DNA Sequencing:

  • Next-generation techniques: Sequence by synthesizing a complementary strand while electronically identifying which nucleotide is added.

  • Third-generation (Nanopore): Identifies nucleotides by moving a single DNA strand through a tiny pore in a membrane and measuring interruptions in electrical current.

Editing Genes and Genomes: CRISPR-Cas9

• CRISPR-Cas9 System: A powerful gene-editing tool discovered with contributions by Jennifer Doudna.

  • Cas9: A nuclease that cuts double-stranded DNA according to a guide RNA complementary to the "target" gene.

  • Applications:

    • Knock out: Disabling a gene to determine its function.

    • Repair: Correcting mutations (e.g., research into correcting sickle-cell disease).

    • Gene Drive: Engineering a new allele to favor its inheritance over the wild-type, rapidly spreading the trait through a population.

Questions & Discussion

• Self-Assessment: Enzyme and Protein Function Identification:

  • Helicase: Unwinds the DNA double helix at the replication fork.

  • Topoisomerase: Relieves DNA supercoiling ahead of the replication fork.

  • Single-stranded binding protein: Binds to and stabilizes single-stranded DNA.

  • Primase: Creates RNA primers which act as a starting point for DNA synthesis.

  • DNA Polymerase III: Builds a new DNA strand using the old DNA strand as a template (primary elongation).

  • DNA Polymerase I: Replaces RNA primers with DNA.

  • DNA Ligase: Covalently joins together Okazaki fragments in the lagging DNA strand and seals gaps in repair processes.