Replication of DNA (Chapter 16)

Replication of DNA (Chapter 16)

Introduction

• Focuses on the mechanisms of DNA replication.

How Does DNA Get Replicated?

• Description of the fundamental process of DNA replication.

Structural Overview of DNA

• DNA structure features:

  • 5′ and 3′ ends:

  • 5′ end: The end of the DNA molecule where a phosphate group is attached.

  • 3′ end: The end of the DNA molecule where a hydroxyl (OH) group is attached.

  • Base Pairing: Each base pair (adenine-thymine: A-T, cytosine-guanine: C-G) is situated 0.34 nm apart.

  • Double Helix Structure: 10 base pairs correspond to 1 complete turn of the helix, which measures 3.4 nm in length.

  • Diameter of DNA Strand: Approximately 2 nm.

Parental Molecule

• Example sequence of a parental DNA strand:

  • 5′ - A C T A G T G A T C - 3′

  • 3′ - T G A T C A C T A G - 5′

• This represents an antiparallel structure of complementary strands.

Separation of Parental Strands into Templates

• Process whereby parental strands separate, creating templates for replication:

  • Initial Parent Strand:

  • 5′ - A C T A G T G A T C - 3′

  • 3′ - T G A T C A C T A G - 5′

  • Resulting in the creation of two template strands for new synthesis.

Formation of New Strands Complementary to Template Strands

• Semi-conservative replication: Each new DNA molecule consists of one parental and one newly synthesized strand.

• Example sequences for resulting new strands during replication:

  • Complementary synthesis to templates:

  • New synthesis will occur in 5′ to 3′ direction only.

Direction of DNA Synthesis

• 5′ to 3′ Direction: DNA synthesis can only occur in this specified direction.

• Requirement of a short RNA primer to initiate synthesis:

  • Primase synthesizes RNA primers complementary to the template strand.

Incoming Nucleotides During Synthesis

• Example base pairing and incoming nucleotides:

  • During synthesis, incoming nucleotides pair with template nucleotides:

  • New strand base: C G T G C A C T

  • Template strand base (3′-5′): A C G

Key Enzymes Involved in DNA Replication

• Helicase: Unwinds the DNA helix, creating replication forks.

• Topoisomerase: Relieves the tension created ahead of the replication fork.

• Primase: Synthesizes RNA primers for both leading and lagging strands.

• DNA Polymerase III (DNA pol III): Main enzyme for DNA synthesis; synthesizes leading strand continuously and lagging strand in fragments (Okazaki fragments).

• DNA Polymerase I (DNA pol I): Replaces RNA primers with DNA nucleotides.

• DNA Ligase: Joins Okazaki fragments to form a continuous DNA strand.

Replication Fork and Bubble Structure

• Origin of Replication in E. coli:

  • DNA replication begins at specific locations (origins) and creates two daughter molecules:

  • Replication Fork: The region where the double-stranded DNA is unwound.

  • Replication Bubble: Characteristic structure formed as replication proceeds, with separate strands being unwound.

Elongation of the Leading Strand

• Continuous synthesis occurs in the 5′ to 3′ direction:

  • DNA pol III adds nucleotides continuously as the fork progresses.

  • Function of single-strand binding proteins: Prevent re-annealing of separated strands.

Elongation of the Lagging Strand

• Lagging Strand Synthesis: Occurs in fragments, known as Okazaki fragments:

  • Primase synthesizes an RNA primer for each fragment.

  • DNA pol III synthesizes each Okazaki fragment separately, then detaches.

Completion of Lagging Strand

• DNA pol I replaces RNA primers for Okazaki fragments:

  • RNA primer for Fragment 2 replaced with DNA.

• DNA Ligase: Forms covalent bonds between DNA fragments to form a complete lagging strand.

Overall Summary of Processes in DNA Replication

• DNA pol III synthesizes the leading strand continuously from the primer.

• Lagging strand synthesized in Okazaki fragments:

  • Each fragment initiated by an RNA primer laid down by primase.

  • After synthesis, RNA primers are replaced with DNA and fragments joined by DNA ligase.