4. Replication of DNA. Necessary elements and mechanism. Replication of linear DNA molecules. Fidelity of replication.

DNA replication.

• DNA replication is essential for life, allowing organisms to reproduce at molecular, cellular, and organismal levels.
• It involves template-based synthesis, where the complementary nature of nucleotides enables accurate copying.
• The double helix structure of DNA naturally suggests a copying mechanism.
• Replication is semi-conservative—each new double helix has one old (template) strand and one new strand.
• The Meselson-Stahl experiment (1958) confirmed this model using isotopic labeling of nitrogen in bacterial DNA.

Mechanism:

1. Strands separate at the origin.
2. Free nucleotides spontaneously base-pair with exposed bases.
3. DNA polymerase forms phosphodiester bonds, linking nucleotides into a new strand.
4. Result: two identical DNA molecules.

Directionality of Synthesis:

• DNA is synthesized 5’ to 3’, using triphosphate nucleotides.
• The template is read 3’ to 5’.
• DNA polymerase adds nucleotides by attaching the new base to the 3’ OH of the growing strand, releasing pyrophosphate.

Phases of Replication:

1. Initiation:

• Begins at replication origins (ori)—usually AT-rich palindromic sequences.
• Prokaryotes: usually one ori. Eukaryotes: multiple origins per chromosome.
• DNA polymerase cannot start a new strand due to its proofreading mechanism.
• Primase, an RNA polymerase, synthesizes short RNA primers (~10 nt) to initiate synthesis.
• Eukaryotic primase is part of a complex with DNA polymerase alpha, which extends the primer to ~40 nt (RNA + DNA).
• The origin is first recognized by a replication initiator protein (e.g., ORC in eukaryotes), which recruits:
• Helicase (unwinds DNA),
• Primase and polymerase,
• Forming a replisome (functional replication unit).
• Helicase is loaded by a helicase loader and becomes part of both the pre-replicative complex and the replisome.

2. Elongation:

• DNA synthesis occurs at the replication fork, a Y-shaped structure.
• Leading strand: synthesized continuously toward the fork.
• Lagging strand: synthesized discontinuously away from the fork in Okazaki fragments (1000–2000 nt in prokaryotes, 100–200 nt in eukaryotes).

3. Termination:Involves primer removal, fragment joining, and resolving end replication problems (for linear DNA).