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:
Strands separate at the origin. Free nucleotides spontaneously base-pair with exposed bases. DNA polymerase forms phosphodiester bonds, linking nucleotides into a new strand. 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:
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 synthesizes short RNA primers (~10 nt) to initiate synthesis.
- Eukaryotic primase works with DNA polymerase alpha to extend primers to ~40 nt (RNA + DNA).
- The origin is recognized by initiator proteins (e.g., ORC), recruiting helicase, primase, and polymerase to form the replisome.
- Helicase is loaded by a helicase loader and becomes part of the pre-replicative complex and replisome.
Elongation:
- DNA synthesis occurs at the replication fork.
- Leading strand: synthesized continuously toward the fork.
- Lagging strand: synthesized discontinuously as Okazaki fragments (1000–2000 nt in prokaryotes, 100–200 nt in eukaryotes).
Termination:
- Primer removal, Okazaki fragment joining, and resolving end-replication issues in linear DNA.