Chapter 12: DNA Replication

Key Concepts

• Semi-conservative Replication: Each strand of the original DNA serves as a template for the formation of a complementary strand, ensuring that half of the parent DNA is retained in each daughter molecule. This mechanism allows for genetic continuity as it preserves the original sequence through generations of cell division.

• Origin of Replication: The specific sequence of DNA where replication begins. In prokaryotes, it usually consists of a single defined site, while eukaryotes have multiple origins to optimize the speed and efficiency of replication due to their larger genome size.

• Leading and Lagging Strands:

  • Leading Strand: Synthesized continuously in the same direction as the unwinding DNA, allowing it to be replicated in a smooth, uninterrupted manner.

  • Lagging Strand: Synthesized discontinuously; formed in short sections known as Okazaki fragments, which are later joined together. The lagging strand is synthesized in the opposite direction of the replication fork movement, necessitating multiple RNA primers.

• DNA Polymerases: Enzymes responsible for adding nucleotides during DNA synthesis. Key activity involves creating phosphodiester bonds between nucleotides to form the new strand. Different types of DNA polymerases have various roles, including some that also facilitate repair processes.

• Telomerase: An enzyme that extends the telomeres of chromosomes, which are crucial for maintaining chromosome integrity during replication. Telomerase adds repetitive nucleotide sequences (TTAGGG in humans) to the ends of chromosomes, delaying their shortening during cell division, which is significant in stem cells and certain types of cancer cells.

Models of DNA Replication

• Conservative Model: The original DNA helix remains unchanged and a completely new double helix is formed. This model was quickly ruled out as it did not fit experimental data.

• Dispersive Model: The original DNA strands are broken into pieces, with new DNA synthesized and reassembled as a hybrid, which also did not match the findings from experimental data.

• Semi-conservative Model (Accepted): The original strand remains intact and pairs with a newly synthesized strand, which was confirmed by the Meselson-Stahl experiment.

Equilibrium Density Gradient Centrifugation - Meselson and Stahl Experiment

• Isotopes: Nitrogen isotopes 14N (light) and 15N (heavy) are used to label DNA, allowing differentiation based on density.

• Density Separation: Different molecules can be separated based on their density during centrifugation, helping to visualize the replication process.

Experimental Predictions

• Conservative: Two separate bands after the first replication (old and new).

• Dispersive: A single band after the first replication, representing a mix of old and new.

• Semi-conservative: An intermediate band after the first replication, then two bands after the second replication showing light and intermediate weights.

Results Supporting the Semi-Conservative Model

• After one replication, a single band appears at an intermediate weight, confirming mixing of old and new DNA. After a second replication, there are two distinct bands showing light and intermediate weights, providing conclusive evidence for the semi-conservative mechanism.

Modes of Replication

• Bacterial Replication:

  • Circular Chromosome: Bacteria typically have a single circular chromosome, which has a single origin of replication (ori).

  • Bidirectional Replication: DNA strands can replicate in a bidirectional manner, forming a replication bubble that facilitates quick duplication of the genetic material.

• Eukaryotic Replication:

  • Linear Chromosomes: Eukaryotic cells have multiple origins of replication along their linear chromosomes. This is necessary due to the larger size of their genomes and the need for rapid replication.

Replication Mechanism

Initiation

• Initiator Proteins: These proteins bind to DNA at the origin of replication, marking the starting point for replication.

• Unwinding:

  • Helicase: Unwinds the DNA strands, creating the replication fork.

  • Single-Stranded Binding Proteins: These proteins bind to the unwound DNA strands to prevent them from re-annealing before they can be replicated.

  • DNA Gyrase: Relieves tension and prevents supercoiling ahead of the replication fork, ensuring smooth progression during unwinding.

Elongation

• DNA Polymerase III: Adds nucleotides to the growing DNA strand in the 5’ to 3’ direction. It cannot initiate synthesis without a primer, which is critical for the start of nucleotide addition.

• Primase: Synthesizes a short RNA primer at the replication fork, providing a free 3' OH group for DNA polymerases to extend.

Lagging Strand Synthesis

• Occurs in fragments known as Okazaki fragments because it runs opposite to the movement of the replication fork. Each fragment starts with a new RNA primer, making the replication process disjointed but effective.

Fragment Joining

• DNA Polymerase I: Replaces RNA primers with DNA nucleotides, ensuring the integrity of the newly synthesized strands.

• DNA Ligase: Seals gaps between fragments, completing the continuous strand and ensuring that the DNA molecule is intact.

Fidelity of Replication

• The error rate for DNA replication is very low (less than one mistake per billion nucleotides), highlighting the precision of cellular machinery.

• Proofreading by DNA Polymerase III: This enzyme corrects errors during synthesis using 3’ to 5’ exonuclease activity, significantly reducing the likelihood of mutations.

• Mismatch Repair Enzymes: Additional enzymes correct any remaining errors post-replication, preserving genetic information.

Telomeres and Aging

• Telomeres: Repetitive nucleotide sequences at both ends of linear chromosomes that protect them from degradation during replication. They play a vital role in the lifespan of cells.

• Telomerase: Extends the telomeres; crucial for cells that divide often (e.g., stem cells) to avoid loss of genetic information over successive divisions.

• Aging and Cancer: While telomerase could extend cell lifespan, it is also linked to most cancer cells allowing unlimited division, contributing to tumorigenesis.

Homework Questions

1, 2, 6, 7, 8, 10, 11, 15, 19, 21*, 22*, 24*, 30, 33*