11.2 DNA Replication - Microbiology | OpenStax

Learning Objectives

• By the end of this section, you should be able to:

  • Explain the meaning of semiconservative DNA replication

  • Describe bidirectional DNA replication, including leading and lagging strands

  • Explain the formation of Okazaki fragments

  • Outline the process of DNA replication and the roles of different enzymes

  • Contrast DNA replication in bacteria and eukaryotes

  • Understand rolling circle replication

Semiconservative DNA Replication

• Semiconservative replication: Each double-stranded DNA molecule consists of one parental and one newly synthesized strand.

• Other competing models included:

  • Conservative model: Parent strands remain associated in one DNA molecule, new strands in another.

  • Dispersive model: Resulting strands contain sections of old and new DNA.

• Watson and Crick's work in 1953 hinted at the replication mechanism by demonstrating how strands separate to serve as templates.

Meselson and Stahl Experiment (1958)

• Experiment with E. coli using heavy nitrogen (N15) to label parental DNA.

• Growth in lighter nitrogen (N14) showed:

  • After one generation, a single band positioned between N15 and N14, indicating semiconservative or dispersive replication.

  • After a second generation, formation of two bands confirmed semiconservative replication since it demonstrated parental and new strands.

• Conclusion: DNA replication is semiconservative, with each original strand serving as a template for new strand synthesis.

DNA Replication in Bacteria

• E. coli: 4.6 million base pairs, replicated in about 42 minutes from a single origin (oriC).

• DNA polymerase Types:

  • DNA pol III: Main enzyme for DNA synthesis.

  • DNA pol I and II: Primarily involved in DNA repair.

Initiation

• Replication begins at origin of replication.

• Proteins binding: Create single-stranded regions for replication.

• Topoisomerase II (gyrase): Relaxes supercoiling.

• Helicase: Opens DNA strands by breaking hydrogen bonds, forming replication forks.

Elongation

• Leading strand: Synthesized continuously towards the replication fork.

  • Extended directly from a single RNA primer.

• Lagging strand: Synthesized discontinuously in Okazaki fragments.

  • Each fragment requires a new RNA primer.

  • Okazaki fragments are separated by RNA primers, synthesized by RNA primase.

Termination

• After replication, circular chromosomes are interlocked (concatenated).

• Topoisomerase IV: Separates interlocked chromosomes and reseals them.

Key Enzymes in Bacterial DNA Replication

DNA Replication in Eukaryotes

• Much larger and more complex than prokaryotes with multiple linear chromosomes (e.g., human genome: 3 billion base pairs).

• Rate of replication: Approximately 100 nucleotides per second (10x slower than prokaryotes).

• Multiple origins of replication per chromosome (30,000 to 50,000 in humans).

• Process includes protein recruitment, similar to the prokaryotic process.

Eukaryotic Specifics

• The leading strand: Continuous synthesis by DNA polymerase δ.

• The lagging strand: Synthesized by polymerase ε, using multiple primers.

• Telomeres: Protect the ends of linear chromosomes, preventing loss of coding sequences over successive divisions.

• Telomerase: Enzyme active in germ and stem cells, maintains chromosome ends by adding repetitive sequences.

Comparison: Bacterial vs Eukaryotic Replication

Rolling Circle Replication

• Used by certain plasmids and some viruses.

• Begins with nicking one strand of circular DNA, allowing unidirectional replication and displacement of the nicked strand.

• New strand forms, eventually creating double-stranded DNA identical to the original.