12 B DNA Replication Review

Learning Goals

• Understand the polarity of DNA replication.
• Describe the steps and key players in DNA replication.

Historical Background of DNA Replication Models

• Semi-Conservative Method: Proposed by Watson and Crick. Each new DNA molecule consists of one old and one new strand.
• Conservative Method: Proposed by Meselson and Stahl, stating that the entire DNA molecule remains intact and a completely new molecule is synthesized.
• Dispersive Method: Proposed by Julian Huxley, suggesting that DNA strands are broken into pieces and reassembled during replication.
• Experimental Evidence: Meselson and Stahl (1958) used cesium chloride centrifugation to confirm the semi-conservative model. They grew E. coli in heavy nitrogen ($^{15}N$) for many generations and then transferred it to lighter nitrogen ($^{14}N$) for further replication, allowing observation of DNA density changes over generations.

Mechanisms of DNA Replication

• Bidirectional Replication in Bacteria: Begins at a single origin of replication and creates replication forks that move in both directions.
• Multiple Origins in Eukaryotes: Eukaryotic DNA replicates at multiple origins within each chromosome.
  • John Cairns (1963): Provided evidence for bacteria's origins of replication.
  • Replication Bubble: Formed as DNA unwinds, characterized by replication forks at each end.

Key Players in DNA Replication

• DNA Polymerase in E. coli: Efficiently adds approximately 1,000 nucleotides per second.
• Enzymes Involved:
  • Replication Initiating Enzymes: Bind and initiate replication at specific consensus sequences at the origin (e.g., OriC).
  • DNA A: First to bind, causing the DNA to bend and break hydrogen bonds.
  • DNA B: Functions as helicase unwinding the DNA.
  • DNA C: Aids in delivering DNA B.
  • Single-Stranded Binding Proteins (SSB): Prevent DNA strands from re-annealing.
  • DNA G (Primase): Synthesizes RNA primers necessary for DNA polymerase to initiate DNA synthesis.
  • DNA Polymerase I: Removes RNA primers and replaces them with DNA, utilizing both exonuclease and polymerase activities.
  • DNA Ligase: Joins Okazaki fragments on the lagging strand, sealing nicks in the DNA.

Directionality in DNA Synthesis

• 5' to 3' Direction: DNA strands are synthesized from the 5' end to the 3' end, affecting the leading and lagging strands:
  • Leading Strand: Synthesized continuously toward the replication fork.
  • Lagging Strand: Synthesized discontinuously in Okazaki fragments away from the replication fork.

DNA Replication Process Steps

1. Helicase Activity: DNA B unwinds the DNA, breaking hydrogen bonds using ATP.
2. Topoisomerase Activity: Relaxes DNA supercoiling caused by unwinding.
3. Synthesis of RNA Primers: Primase synthesizes RNA primers, enabling DNA polymerase to continue synthesis.
4. DNA Synthesis: DNA polymerases add nucleotides to the 3' end of the primers, forming new DNA strands.
5. Removal of RNA Primers and Gap Filling: DNA Polymerase I removes RNA primers, fills gaps with DNA nucleotides, while Ligase seals fragments.

Proofreading and Accuracy

• Proofreading Activity: DNA polymerases have 3' to 5' exonuclease activity to correct errors by excising mismatched nucleotides.
• Error Rate: Approximately one error per billion nucleotides during replication.

Eukaryotic vs. Prokaryotic DNA Replication

• Eukaryotic Replication: Involves multiple polymerases (alpha, epsilon, and delta) and requires a sliding clamp (PCNA) for efficient replication.
• Clamp Loader and Sliding Clamp: Ensures that DNA polymerase remains attached to the template strand during synthesis.

Telomeres and Telomerase

• Telomeres: Repetitive sequences at chromosome ends protect vital genes during DNA replication.
• Telomerase Function: Synthesizes telomere repeats, compensating for incomplete replication at chromosome ends, primarily active in germline cells and some stem cells.
• Clinical Implications: Inactive telomerase in differentiated somatic cells leads to limited cell divisions; reactivation can contribute to aging and cancer, such as in Werner syndrome, which accelerates aging characteristics.

Conclusion

• DNA replication is a complex but highly regulated process involving various enzymes; understanding its mechanisms is crucial for studying cellular division and genetics. The principles remain similar between prokaryotes and eukaryotes despite differences in the enzymes involved.