DNA Structure, Replication, and Packaging: Study Notes for Cell Biology

DNA Structure

Nucleotide Structure

The basic unit of DNA is the nucleotide, which consists of a deoxyribose sugar, a phosphate group, and a nitrogenous base. Nucleotides are linked together by phosphodiester bonds to form the DNA backbone.

• Deoxyribose sugar: Numbered carbons (1', 2', 3', 4', 5') indicate the orientation and polarity of the DNA strand (5' to 3').

• Phosphate group: Attached to the 5' carbon of the sugar; forms the backbone of the DNA strand.

• Nitrogenous base: Adenine (A), Thymine (T), Cytosine (C), Guanine (G); bases pair via hydrogen bonds (A-T, C-G).

• Polarity: DNA strands run antiparallel; one strand 5'→3', the other 3'→5'.

Example of complementary base pairing:

• A pairs with T via 2 hydrogen bonds.

• C pairs with G via 3 hydrogen bonds.

Bond comparison:

• Hydrogen bonds: Between bases, hold the two DNA strands together.

• Phosphodiester bonds: Between the 3' carbon of one sugar and the 5' phosphate of the next; form the backbone.

Key Experiments in DNA Structure

• Chargaff's Rules: Amount of A = T, C = G in DNA; base pairing is specific.

• Griffith's Experiment: Demonstrated transformation in bacteria (S and R strains of Streptococcus pneumoniae).

• Avery, MacLeod, McCarty: Identified DNA as the transforming principle.

• Hershey-Chase Experiment: Used bacteriophage to show DNA, not protein, is genetic material.

Application: These experiments established DNA as the molecule of heredity and clarified its structure.

DNA Replication

Overview and Replication Rates

DNA replication is the process by which a cell copies its DNA before cell division. It is semi-conservative, meaning each new DNA molecule contains one old and one new strand.

• Replication rate in E. coli: ~1000 nucleotides/second.

• Replication rate in Homo sapiens: ~50 nucleotides/second.

• Human genome size: $3 \times 10^9$ base pairs.

• Replication origins: Humans have thousands of replication origins per genome to ensure timely replication.

Meselson-Stahl Experiment

This experiment demonstrated the semi-conservative nature of DNA replication using isotopic labeling and density gradient centrifugation.

• Hypothesis: DNA replicates semi-conservatively.

• Experimental design: Grow bacteria in heavy nitrogen ($^{15}N$), then switch to light nitrogen ($^{14}N$); analyze DNA after replication.

• Results: After one round, DNA was intermediate density; after two rounds, both intermediate and light DNA were present.

• Main finding: Each daughter DNA molecule contains one parental and one newly synthesized strand.

DNA Replication Mechanism

Replication begins at origins and proceeds bidirectionally, forming replication forks. Key enzymes coordinate the process.

• Helicase: Unwinds the DNA double helix.

• Single-stranded binding proteins: Stabilize unwound DNA.

• Primase: Synthesizes short RNA primers.

• DNA polymerase III (prokaryotes): Extends DNA from the primer.

• DNA polymerase I: Removes RNA primers and fills gaps with DNA.

• DNA ligase: Seals nicks between Okazaki fragments on the lagging strand.

• DNA gyrase/topoisomerase: Relieves supercoiling ahead of the fork.

Leading vs. Lagging Strand:

• Leading strand: Synthesized continuously in the 5'→3' direction.

• Lagging strand: Synthesized discontinuously as Okazaki fragments, each requiring a new primer.

Replication fork diagram: Shows directionality, enzyme locations, and primer placement.

Steps in DNA Replication (Order)

1. DNA unwinds at the origin of replication.

2. Single-strand binding proteins coat DNA to prevent rewinding.

3. Topoisomerase/gyrase relieves supercoiling ahead of the fork.

4. Primase synthesizes RNA primers.

5. Helicase opens up the DNA, forming replication forks.

6. DNA polymerase III extends the new DNA strand from the primer.

7. Elongation of both leading and lagging strands continues.

8. RNA primers are removed by exonuclease activity (DNA polymerase I).

9. DNA polymerase I fills gaps with DNA.

10. DNA ligase seals nicks between fragments, forming phosphodiester bonds.

Replication in Circular vs. Linear DNA

• Circular DNA (e.g., bacteria): Replication proceeds around the circle; no ends, so no shortening.

• Linear DNA (eukaryotes): Ends (telomeres) can shorten with each replication; special mechanisms (telomerase) maintain length.

DNA Replication in Plant Cells: Experimental Analysis

Pulse-labeling with radioactive thymidine allows tracking of newly synthesized DNA strands through cell cycles.

• Cells labeled at T=0, analyzed at T=8h and T=24h after mitosis.

• Autoradiographs show distribution of labeled and unlabeled DNA strands in chromatids.

• Results support semi-conservative replication: after one division, chromatids have one labeled and one unlabeled strand; after two, some chromatids have only unlabeled strands.

DNA Packaging

Chromatin Structure and Nucleosomes

DNA in eukaryotic cells is packaged into chromatin, which allows efficient storage and regulation.

• Nucleosome: Fundamental unit of chromatin; DNA wrapped around histone protein core.

• "Pearls on a string" (1974): Visualization of nucleosomes; 6:1 packing ratio of DNA around chromatin.

• Histone complexes (1975): Histones form complexes for DNA packaging.

• Higher-order structure (1976, 1986): Nucleosomes coil to form more compact structures; multiple layers of DNA compaction.

Application: Chromatin structure regulates gene expression and protects DNA integrity.

Practice and Review Questions

DNA Sequence and Complementarity

Given a DNA sequence, the complementary strand is formed by pairing A with T and C with G, and indicating the correct polarity.

• Example: 5'-TGAGGATCGGGGTTAGTCTTAGACTCCCTTATGCGAARACTT-3'

• Complementary: 3'-ACTCCTAGCCCCAATCAGAATCTGAGGGAATACGCTTTTGAA-5'

Replication Fork Annotation

At the replication fork, students should be able to:

• Identify leading and lagging strands.

• Place RNA primers and Okazaki fragments.

• Indicate where DNA polymerases, helicase, gyrase, and ligase act.

Enzyme Functions and Consequences

• If primase is absent, no RNA primers are made; DNA polymerase cannot initiate synthesis.

• If ligase is absent, Okazaki fragments remain unconnected; lagging strand is incomplete.

• If helicase is absent, DNA cannot unwind; replication stalls.

Summary Table: DNA Replication Enzymes and Functions

Enzyme	Function
Helicase	Unwinds DNA double helix
Single-stranded binding protein	Stabilizes unwound DNA
Primase	Synthesizes RNA primers
DNA polymerase III	Extends DNA from primer (main synthesis)
DNA polymerase I	Removes RNA primers, fills gaps with DNA
DNA ligase	Seals nicks between fragments
Topoisomerase/Gyrase	Relieves supercoiling ahead of fork

Additional info:

• DNA replication is tightly regulated and occurs during the S phase of the cell cycle.

• Errors in replication can lead to mutations, which may cause disease.

• Multiple origins of replication in eukaryotes allow rapid duplication of large genomes.