DNA Replication Mechanisms

DNA Polymerase Functions

• DNA polymerase I removes RNA primers and fills gaps using 5’-3’ exonuclease activity, allowing for proofreading.
• DNA polymerases II, IV, and V are involved in repairing damage caused by external forces like UV light.

DNA Polymerase III Holoenzyme

• Composed of two core enzymes, a sliding clamp loader, and a sliding clamp to enhance processivity.
• Major enzyme complex for prokaryotic DNA replication, adding nucleotides to RNA primers and synthesizing leading and lagging DNA strands.
• Enzymes and proteins at the replication fork form a structure known as the replisome.

Mechanism of DNA Synthesis

• DNA replication is bidirectional but occurs biochemically in one direction (5' to 3').
• Single-stranded binding proteins prevent reformation of hydrogen bonds.
• Gyrase alleviates tension from unwinding DNA.
• Primase provides RNA fragments necessary for DNA polymerase III to start polymerization.
• Leading strand synthesis is continuous; lagging strand synthesis involves Okazaki fragments and is discontinuous.
• Lagging strand gaps are filled and ligated post-replication.

Eukaryotic DNA Synthesis

• Requires deoxyribonucleoside triphosphates (dNTPs), template, and primer.
• More complex due to increased DNA volume and linear chromosomes, with DNA dissociating from histones before replication.
• Synthesis of DNA and production of histones occurs simultaneously, doubling histone count during replication.

Comparison: Prokaryotic vs. Eukaryotic Replication

• Prokaryotes have a single origin of replication; eukaryotes have multiple.
• Prokaryotes expand from a single replication loop; eukaryotes have multiple loops for increased efficiency.
• Prokaryotic replication ends at a single termination site; eukaryotic involves merging two replication forks and telomere replication.

Eukaryotic DNA Polymerases

• Pol α initiates and synthesizes the RNA primer, adds 10-20 nucleotides, and is replaced by Pol δ and Pol ε for further synthesis.
• Pol δ synthesizes the lagging strand; Pol ε synthesizes the leading strand.
• Pol γ is responsible for mitochondrial DNA replication.

Telomeres and Aging

• Linear chromosome ends, known as telomeres, shorten with repeated cell divisions in somatic cells lacking telomerase.
• Telomeres contain repetitive G-rich sequences and do not code for genes.
• TERT gene encodes telomerase reverse transcriptase crucial for telomere maintenance.
• TERC component serves as a guide for enzyme attachment and as a template for DNA synthesis.

DNA Recombination

• Occurs through reciprocal exchange of homologous chromosomes involving several steps:
  • Endonuclease nicks DNA strands.
  • Strand displacement pairs loose ends with complementary sequences.
  • Ligation forms hybrid heteroduplex DNA.
• Cross-bridged configurations move down chromosomes via branch migration, resulting in longer heteroduplex regions.
• Formation of the chi-form/Holliday structure precedes additional endonuclease nicking and final ligation, completing recombinant duplex formation.
• Key protein involved is RecA in homologous recombination.