DNA Replication: Enzymes, Mechanisms, and Molecular Analysis

DNA Replication: Enzymes and Mechanisms

Primer Removal and Okazaki Fragment Ligation

During DNA replication, the synthesis of the lagging strand produces short DNA fragments known as Okazaki fragments. The removal of RNA primers and the joining of these fragments are essential for producing a continuous DNA strand.

• DNA polymerase I (pol I) in E. coli uses two main activities:

  • 5'-3' exonuclease activity: Removes RNA primers from the newly synthesized DNA.

  • 5'-3' polymerase activity: Adds DNA nucleotides to the 3' end of the preceding DNA segment, filling in the gap left by primer removal.

• DNA ligase: Seals the remaining nicks between DNA fragments by forming phosphodiester bonds, resulting in a continuous DNA strand.

Example: In E. coli, after the RNA primer is removed by pol I, DNA ligase joins the Okazaki fragments to complete the lagging strand.

Removal and Replacement of RNA Primer Nucleotides in E. coli

The process of removing RNA primers and ligating Okazaki fragments involves several coordinated steps:

1. DNA pol I binds to the single-stranded gap between DNA and the RNA primer.

2. Pol I removes each RNA primer nucleotide using its 5'-3' exonuclease activity.

3. Pol I fills the gap with DNA nucleotides using its 5'-3' polymerase activity.

4. When primer removal is complete, DNA ligase replaces pol I at DNA-DNA single-stranded gaps.

5. DNA ligase catalyzes the formation of a phosphodiester bond to join Okazaki fragments.

Properties of DNA Polymerases

Selected Bacterial, Eukaryotic, and Archaeal DNA Polymerases

DNA polymerases are essential enzymes for DNA replication, repair, and proofreading. Their properties and functions vary among bacteria, eukaryotes, and archaea.

Bacterial Polymerase	Functions
Primase	RNA primer synthesis
DNA polymerase I	RNA primer removal, proofreading, mutation repair
DNA polymerase III	DNA replication, proofreading

Eukaryotic Polymerase	Functions
Pol α	Primer synthesis and lagging strand synthesis
Pol δ	Lagging strand synthesis, proofreading, DNA mutation repair
Pol ε	Leading strand synthesis, proofreading, DNA mutation repair

Archaeal Polymerase	Functions
Primase	Primer synthesis
Pol B	DNA synthesis
Pol D	DNA synthesis

Synthesis of Leading and Lagging Strands at the Replication Fork

Coordination of DNA Synthesis

At the replication fork, both leading and lagging strands are synthesized simultaneously by a complex called the replisome. The processivity of DNA polymerases is enhanced by accessory proteins.

• Sliding clamp: A protein complex that encircles DNA and anchors DNA polymerase to the template, increasing the enzyme's processivity.

• The sliding clamp forms a ring ("doughnut hole") around the DNA, allowing rapid and efficient DNA synthesis.

Example: The sliding clamp in E. coli is the β-clamp, which is essential for high-speed DNA replication.

Additional info:

• The leading strand is synthesized continuously in the 5' to 3' direction, while the lagging strand is synthesized discontinuously as Okazaki fragments.

• Accessory proteins such as the clamp loader help assemble the sliding clamp onto DNA.