DNA Replication: Process and Rules

• DNA replication outcomes should include describing the semiconservative model, understanding essential replication terms, comparing leading and lagging DNA strands, and explaining the role of DNA polymerases.
• DNA must carry information, must replicate itself, allow for change via mutation, and govern the expression of the phenotype via gene function.
• Accurate DNA replication preserves the genome's integrity across generations.
• Replication in eukaryotes occurs during the S phase of the cell cycle.
• Eukaryotic replication is slower but has higher fidelity/accuracy because there is DNA repair systems

Basic Rules of Replication

• DNA replication follows a semi-conservative mechanism.
• Replication occurs at specific sites called replicons.
• Replication is bidirectional, proceeding in both directions from the origin.
• Replication is semi-discontinuous, with both leading and lagging strands synthesized differently.
• Synthesis always occurs in the 5'-3' direction.
• RNA priming initiates DNA synthesis.

Semi-Conservative Mechanism

• DNA replication produces two daughter double helices, each with one conserved strand and one newly synthesized strand.

DNA Replication Models

• In the semiconservative model, each parent strand serves as a template for a new daughter strand.
• In the dispersive model, each generation of daughter DNA contains a mixture of parental and newly synthesized DNA.
• In the conservative model, the first replication yields the original parent double helix and an entirely newly replicated molecule.

Meselson and Stahl Experiments

• Meselson and Stahl grew cells in media containing 14N and 15N to create light and heavy DNA.
• They transferred cells grown in heavy medium to light medium.
• DNA was treated with a high concentration of cesium chloride used density gradient centrifugation techniques.
• Meselson and Stahl's data aligned with the semiconservative model.
• The first replication in 14N medium produced a band of hybrid (15N-14N) DNA.
• A second replication produced both light and hybrid DNA.
• They concluded that replication follows the semiconservative model.

Initiation of Replication

• Initiator proteins recognize specific base sequences on DNA, marking the replication origin.
• Prokaryotic genomes feature a single, circular DNA with one replicon.
• Prokaryotes have a single origin site.
• Eukaryotic genomes have multiple linear chromosomes and replicons.
• Eukaryotes have multiple sites of origin.
• Prokaryotic chromosomes and many bacteriophage and viral DNA molecules are circular with a single replicon.
• A single termination site sits roughly 180° from the unique origin in prokaryotes.
• Linear viral DNA molecules typically have a single origin of replication.
• Eukaryotic replicons and replication bubbles are multiple.
• Eukaryotic chromosomes are long, linear DNA molecules with multiple regions, each featuring its own origin.
• Replication forks from adjacent replication bubbles merge, forming completely replicated DNA; distinct termini aren’t required.
• Replication begins at specific sites where parental strands separate and form replication bubbles.
• These bubbles expand as DNA replication proceeds bidirectionally.
• Replication bubbles fuse, completing daughter strand synthesis.

Bidirectional Replication

• Replication forks move bidirectionally from the origin.
• Bidirectional replication involves forks moving in opposite directions, using each strand as a template for a new daughter strand.

Semi-Discontinuous Replication

• Antiparallel strands are replicated simultaneously.
• The leading strand is synthesized continuously in the 5'-3' direction.
• The lagging strand is synthesized in fragments (Okazaki fragments) in the 5'-3' direction, which must then be joined.

DNA Synthesis Direction

• New strand synthesis always proceeds in the 5'-3' direction.

DNA Replication Details

• DNA replication is semidiscontinuous.
• DNA polymerase can only add nucleotides to the 3' hydroxyl group of the growing strand.
• Antiparallel strands are replicated simultaneously.
• The leading strand is synthesized continuously in the 5'-3' direction.
• The lagging strand is synthesized in fragments in the 5'-3' direction.

Synthesis Direction and Speed

• Synthesis always occurs in the 5'-3' direction, ensuring correct base pairing from the template.
• DNA polymerase catalyzes polymerization, copying complementary base pairs.
• The substrate used is dNTP (deoxyribonucleoside triphosphate).
• The forks move rapidly at 1000 nucleotide pairs per second in bacteria.
• The forks move rapidly at 100 nucleotide pairs per second in humans.
• The slower rate in humans is due to difficulties replicating DNA through the complex chromatin structure of eukaryotic chromosomes.

Self-Correcting DNA Polymerase

• If an incorrect nucleotide is added, DNA polymerase cleaves and replaces it with the correct nucleotide before continuing.

RNA Primers

• DNA polymerase can only add nucleotides to the 3' end of a growing DNA strand.
• RNA primase provides a base-paired 3' end, synthesizing ~10 nucleotide primers.
• The first nucleotides of Okazaki fragments are ribonucleotides.
• DNA synthesis is primed by RNA, which is then removed.
• RNA primers ensure high fidelity of replication.