Chapter 14: DNA — Structure and Function

1869 — Friedrich Miescher

• Isolated phosphate-rich material from nuclei of white blood cells.

• Called it nuclein → later known as DNA.

Frederick Griffith (1928) — Transforming Principle

• Used S (smooth, pathogenic) and R (rough, non-pathogenic) strains of S. pneumoniae.

• Heat-killed S + live R → mouse died; R transformed into S.

Avery, MacLeod, McCarty (1944)

• Showed DNA is the substance responsible for transformation.

• Hershey & Chase (1952)

• Used phages labeled with 35S (protein) or 32P (DNA).

• Only 32P entered bacteria → DNA is genetic material.

Erwin Chargaff (1950)

• DNA made of 4 nucleotides: A, T, C, G.

• Chargaff’s rules:

  • %A = %T

  • %C = %G

• Rosalind Franklin, Watson & Crick (1953)

• Franklin’s X-ray diffraction → double helix.

• Watson & Crick modeled DNA structure.

 Structure of Nucleotides

• 3 parts:

  1. Sugar (deoxyribose in DNA, ribose in RNA)

  2. Nitrogenous base (A, T, C, G; U in RNA)

  3. Phosphate group

• Purines = A, G (double ring)

• Pyrimidines = T, C (single ring)

DNA Structure

• DNA = double helix, antiparallel strands.

• Strands held together by hydrogen bonds (A-T, C-G).

• Covalent phosphodiester bonds connect nucleotides.

• Has major and minor grooves → binding sites for proteins.

Covalent phosphodiester bonds

connect nucleotides.

hydrogen bonds

holds strands together (A-T, C-G).

Models of DNA Replication

1. Conservative → original DNA stays intact, new DNA made separately.

2. Semi-conservative → each strand acts as a template for new strand. ✅

3. Dispersive → DNA broken and mixed into new strands.

• Meselson & Stahl proved semi-conservative model using 15N/14N E. coli experiment.

Conservative

original DNA stays intact, new DNA made separately.

Semi-conservative

each strand acts as a template for new strand. ✅

Dispersive

DNA broken and mixed into new strands.

Topoisomerase (DNA) gyrase

prevents overwinding → Relieves coiling ahead of the fork

Single-Strand Binding (SSB) proteins

keep strands apart

Helicase

Unwinds the DNA double helix.

Primase

makes short RNA primers

DNA Polymerase III

builds new DNA strand (5′ → 3′ direction).

DNA Polymerase I

replaces RNA primers with DNA.

DNA Ligase joins Okazaki fragments

continuous strand

DNA Ligase

Seals gaps between Okazaki fragments

What Is DNA Replication

• Process of copying DNA before cell division.

• Each old strand acts as a template to make a new strand.

• Happens in the cytoplasm of prokaryotes (nucleoid region).

How Replication Happens

1. Helicase opens DNA at the origin.

2. SSB proteins stabilize open strands.

3. Primase adds RNA primers.

4. DNA Pol III adds nucleotides to make new DNA.

5. Leading Strand – continuous synthesis (1 primer).

6. Lagging Strand – made in short Okazaki fragments (many primers).

7. DNA Pol I replaces primers with DNA.

8. DNA Ligase connects fragments.

9. DNA Gyrase separates the two circular DNAs at the end.

Proofreading

DNA polymerase fixes mistakes while copying

Mismatch Repair

Wrong base removed and replaced after replication.

Nucleotide Excision Repair

Fixes thymine dimers caused by UV light

Mutations

Happen when errors aren’t corrected.

• Point mutations: silent, missense, nonsense.

• Frameshift mutations: insertions, deletions.

• Chromosome mutations: inversions, duplications, translocations, fusions.

Point mutations

silent, missense, nonsense

Frameshift mutations

insertions, deletions.

Chromosome mutations

inversions, duplications, translocations, fusions.

 Telomerase (Eukaryotes Only)

• Telomerase maintains chromosome ends (telomeres) in eukaryotes.

• Discovered by Elizabeth Blackburn (2009 Nobel Prize).

 Eukaryotes vs. Prokaryotes

Feature	Eukaryote	Prokaryote
Nucleus	Present	None (DNA in nucleoid)
Chromosome Type	Linear	Circular
Telomerase	Yes	No
Origins of Replication	Many	One

DNA Compaction (Eukaryotes)

1. DNA wraps around histones → nucleosomes.

2. Nucleosomes coil into a solenoid.

3. Looped and attached to scaffold proteins.

4. Forms condensed chromosomes.

DNA Sequencing (Sanger Method)

• Uses dideoxynucleotides to stop DNA synthesis.

• Fragments separated by capillary electrophoresis.

• Laser scanner reads DNA sequence (electropherogram).

Capillary electrophoresis

• A technique to separate molecules (like DNA, RNA, or proteins) based on size and charge.

• The sample is placed in a thin tube (capillary) and an electric field is applied.

• Negatively charged molecules (like DNA) move toward the positive end.

• Smaller molecules move faster than larger ones → separation by size.

Gel Electrophoresis

• DNA is negatively charged → moves to positive pole.

• Small fragments move faster through gel.

• Fragments are sorted by size.