DNA Replication Process: Mechanisms and Key Enzymes

Main Characteristics of DNA Replication

• Semiconservative: Each new DNA molecule is formed by an original DNA strand and one newly synthesized strand. Synthesis occurs by the addition of mononucleotides in the 5' to 3' direction.
• Bidirectional: From a single origin, two replication forks move in opposite directions. In bacterial viruses, there is a single starting point, while in eukaryotes, there are several called replicons. Each DNA fragment is replicated from a single origin.
• Semi-discontinuous: In one strand (the leading strand), fragments are synthesized continuously. In the other strand (the lagging strand), synthesis is discontinuous, meaning small fragments are synthesized separately and then joined together. These are known as Okazaki fragments.

Mechanism of DNA Replication: Initiation, Elongation, and Termination

DNA replication achieves two new double helices from a parental DNA double helix. Each of the new molecules is formed by a daughter strand and a parental strand. With this semiconservative synthesis, we obtain two identical double helices, which are also identical to the parental double helix, ensuring that genetic information is maintained invariably. The mechanism of replication of the circular chromosome of E. coli is reasonably well understood. It involves three main stages: initiation, elongation, and termination.

Enzymes Involved in DNA Replication

• Primase (RNA-dependent DNA polymerase): Synthesizes the RNA primer.
• Helicases: Break the hydrogen bonds, unwinding the DNA double helix.
• Topoisomerases: Eliminate general tension in the double helix produced by the unwinding action of helicases.
• SSB Proteins (Single-Strand Binding Proteins): Bind to single strands at sites where the helix has unwound but has not yet been duplicated, stabilizing them during replication. They prevent DNA tangling, leaving the template ready for copying.
• Nucleases: Break phosphodiester bonds between nucleotides.
• Ligases: Join adjacent DNA fragments by forming phosphodiester bonds.
• DNA Polymerase: Catalyzes the formation of phosphodiester bonds between nucleotides, adding complementary deoxyribonucleoside triphosphates (dNTPs). DNA polymerase binds the correct nucleotide based on the geometry of the active site and the incoming nucleotide.

Prokaryotic DNA Polymerases

• DNA Polymerase I: Cleaves the RNA primer and fills the gaps.
• DNA Polymerase II: Primarily involved in DNA repair.
• DNA Polymerase III: The main replication enzyme. It has 5'-3' polymerase activity and 3'-5' exonuclease activity (for proofreading).
• DNA Polymerase IV and DNA Polymerase V: Involved in DNA repair and translesion synthesis.

Eukaryotic DNA Polymerases

• DNA Polymerase α (Alpha): Initiator; synthesizes short DNA stretches after the RNA primer.
• DNA Polymerase β (Beta): Primarily involved in DNA repair in the nucleus.
• DNA Polymerase γ (Gamma): The only polymerase found in mitochondria, responsible for mitochondrial DNA replication.
• DNA Polymerase δ (Delta): Has 5'-3' polymerase and 3'-5' exonuclease activity. It synthesizes the lagging strand.
• DNA Polymerase ε (Epsilon): Has 5'-3' polymerase and 3'-5' exonuclease activity. It synthesizes the leading strand.