The Molecular Basis of Inheritance (Chapter 16): Structure, Function, and Replication of DNA

The Search for the Genetic Material

Historical Experiments Identifying DNA as Genetic Material

The identification of DNA as the genetic material was a major milestone in biology, achieved through a series of key experiments.

• T. H. Morgan's Group (1910): Demonstrated that genes are located on chromosomes, which are composed of DNA and protein. This led to the question of which molecule—DNA or protein—carries genetic information.

• Frederick Griffith (1928): Worked with two strains of Streptococcus pneumoniae: a pathogenic (smooth, S) and a harmless (rough, R) strain. He discovered that mixing heat-killed S cells with living R cells transformed the R cells into pathogenic S cells, suggesting a "transforming principle."

Conclusion: Living R bacteria could be transformed into pathogenic S bacteria by a substance in the heat-killed S cells, indicating the presence of a heritable material.

• Oswald Avery, Maclyn McCarty, and Colin MacLeod (1944): Identified DNA as the transforming substance by systematically eliminating proteins and RNA from extracts of S cells. Only DNA was able to transform R cells into S cells.

Treatment	Result
Protease (removes protein)	Transformation occurs
RNase (removes RNA)	Transformation occurs
DNase (removes DNA)	No transformation

Conclusion: Transformation requires DNA, confirming it as the genetic material.

• Alfred Hershey and Martha Chase (1952): Used bacteriophage T2 to infect E. coli. By labeling phage DNA with radioactive phosphorus (32P) and protein with radioactive sulfur (35S), they showed that only DNA entered the bacterial cells and directed viral replication.

Conclusion: DNA, not protein, is the genetic material in phages.

DNA as the Genetic Material

DNA Structure and Composition

DNA is a polymer composed of nucleotides, each containing a nitrogenous base, a deoxyribose sugar, and a phosphate group.

• Erwin Chargaff (1950): Discovered that DNA composition varies between species and that the amount of adenine (A) equals thymine (T), and the amount of guanine (G) equals cytosine (C). This is known as Chargaff's Rules.

Source	Adenine (%)	Guanine (%)	Cytosine (%)	Thymine (%)
E. coli	24.7	26.0	25.7	23.6
Wheat	28.1	21.8	22.7	27.4
Sea urchin	32.8	17.7	17.3	32.1
Salmon	29.7	20.8	20.4	29.1
Human	30.4	19.6	19.9	30.1
Ox	29.0	21.2	21.2	28.7

Example: In humans, the percentage of A is approximately equal to T, and G is approximately equal to C, supporting the base-pairing model.

Building a Structural Model of DNA

X-ray Crystallography and the Double Helix

The structure of DNA was elucidated through X-ray crystallography and model building.

• Maurice Wilkins and Rosalind Franklin: Used X-ray diffraction to study DNA, revealing its helical structure and regular spacing of bases.

• James Watson and Francis Crick: Built a double helix model consistent with Franklin's data and chemical knowledge. They proposed that DNA consists of two antiparallel sugar-phosphate backbones with nitrogenous bases paired in the interior.

• Base Pairing: Purine (A or G) pairs with pyrimidine (T or C) to maintain a uniform width, consistent with X-ray data. A pairs with T via two hydrogen bonds, and G pairs with C via three hydrogen bonds.

Key Features of DNA Structure:

• Double helix with antiparallel strands (5' to 3' and 3' to 5')

• Complementary base pairing (A=T, G≡C)

• Major and minor grooves for protein binding

DNA Replication

Mechanism of DNA Replication

DNA replication is the process by which a cell copies its DNA before cell division. The double helix unwinds, and each strand serves as a template for a new complementary strand.

• Semiconservative Model: Each daughter DNA molecule consists of one parental strand and one newly synthesized strand.

Key Steps in DNA Replication:

1. Initiation: Replication begins at origins of replication, forming replication bubbles and forks.

2. Unwinding: Helicase unwinds the DNA; single-strand binding proteins stabilize the unwound strands; topoisomerase relieves supercoiling.

3. Priming: Primase synthesizes short RNA primers to provide a starting point for DNA polymerase.

4. Elongation: DNA polymerase adds nucleotides to the 3' end of the primer, synthesizing the new strand in the 5' to 3' direction.

5. Leading and Lagging Strands: The leading strand is synthesized continuously; the lagging strand is synthesized discontinuously as Okazaki fragments, which are later joined by DNA ligase.

Key Enzymes and Proteins:

• Helicase: Unwinds the DNA double helix.

• Single-strand binding proteins: Stabilize unwound DNA.

• Topoisomerase: Relieves tension ahead of the replication fork.

• Primase: Synthesizes RNA primers.

• DNA polymerase III: Main enzyme for DNA synthesis.

• DNA polymerase I: Replaces RNA primers with DNA.

• DNA ligase: Joins Okazaki fragments on the lagging strand.

Example: In E. coli, replication starts at a single origin and proceeds bidirectionally, forming two daughter DNA molecules.

Proofreading and Repair

DNA polymerases proofread newly synthesized DNA, correcting errors. Additional repair mechanisms further reduce the error rate.

• Mutation: Sequence changes that escape repair become permanent and can be inherited. Mutations are the source of genetic variation for evolution.

Chromatin Structure

Organization of DNA in Eukaryotic Cells

In eukaryotes, DNA is packaged with proteins into chromatin, allowing it to fit within the nucleus and regulate gene expression.

• Nucleosome: The basic unit of chromatin, consisting of DNA wrapped around histone proteins ("beads on a string").

• Higher-order Packing: Nucleosomes coil into a 30-nm fiber, which forms looped domains and further condenses into metaphase chromosomes during cell division.

Types of Chromatin:

• Euchromatin: Loosely packed, transcriptionally active.

• Heterochromatin: Densely packed, transcriptionally inactive (e.g., centromeres, telomeres).

Example: During interphase, most chromatin is euchromatin, but some regions remain as heterochromatin, restricting gene expression.

Summary Table: Key Experiments in Identifying DNA as Genetic Material

Scientist(s)	Year	Experiment	Conclusion
Griffith	1928	Transformation in bacteria	Heritable substance can transform cells
Avery, MacLeod, McCarty	1944	Biochemical identification of transforming principle	DNA is the genetic material
Hershey & Chase	1952	Phage labeling with radioactive isotopes	DNA, not protein, is genetic material
Chargaff	1950	Base composition analysis	A=T, G=C; DNA varies by species
Franklin, Wilkins	1952	X-ray diffraction	DNA is helical
Watson & Crick	1953	Model building	Double helix structure

Practice Questions

1. Relate Griffith's experiment to the identification of the genetic material.

2. What important next step did Avery, MacLeod, and McCarty take?

3. What is the importance of Chargaff's discovery?

Additional info: This guide expands on the original notes by providing definitions, context, and examples for each experiment and concept, as well as summarizing the key steps and enzymes involved in DNA replication and chromatin structure.