Overview
This lecture explains the molecular process of DNA replication, covering its purpose, mechanisms, stages, key enzymes involved, and relevant clinical applications.
Purpose and Fundamentals of DNA Replication
- DNA replication ensures each new cell receives an identical copy of genetic information during the S phase of the cell cycle.
- Replication is semi-conservative: each daughter DNA molecule has one old (parental) strand and one newly-synthesized strand.
- DNA synthesis always proceeds in the 5’ to 3’ direction.
- Replication is bi-directional, starting from origins of replication and moving outward at replication forks.
Stages of DNA Replication
- Initiation: Begins at AT-rich origins of replication, which are easier to separate due to fewer hydrogen bonds.
- Multiple origins exist in eukaryotic chromosomes, each bound by a pre-replication protein complex.
- Single-stranded binding proteins (SSBPs) protect unwound DNA strands from re-annealing and nuclease degradation.
- Helicase unwinds DNA using ATP; topoisomerases relieve supercoiling ahead of the fork (type I in eukaryotes does not need ATP; II and IV do).
- Elongation: Primase synthesizes short RNA primers; DNA polymerase III extends DNA from primers in a 5’ to 3’ direction.
- Leading strand synthesized continuously; lagging strand synthesized discontinuously as Okazaki fragments.
- DNA polymerase III also has 3’→5’ exonuclease (proofreading) activity.
- DNA polymerase I replaces RNA primers with DNA and has both 5’→3’ (primer removal) and 3’→5’ (proofreading) exonuclease activities.
- DNA ligase joins DNA fragments, especially on the lagging strand.
Clinical Applications
- Topoisomerase inhibitors (e.g., irinotecan, etoposide for eukaryotes; fluoroquinolones for prokaryotes) block DNA replication in cancer or bacteria.
- Nucleoside reverse transcriptase inhibitors (NRTIs) block HIV replication by lacking 3’ OH, preventing DNA elongation.
Termination and Telomeres
- Replication ends when forks meet and DNA polymerases detach.
- Telomeres are non-coding repetitive DNA at chromosome ends that shorten with each division, preventing loss of coding genes.
- The Hayflick limit is the maximum number of replications before telomere loss affects genes.
- Telomerase enzyme extends telomeres in stem cells and cancer cells via reverse transcription (RNA template to DNA).
Key Terms & Definitions
- Semi-conservative replication — Each new DNA helix contains one old and one new strand.
- Origin of replication — DNA region where replication starts.
- Replication fork — Y-shaped point where DNA is split for replication.
- Okazaki fragments — Short DNA pieces made on the lagging strand.
- Helicase — Enzyme that unwinds DNA.
- Topoisomerase — Enzyme that relieves DNA supercoiling.
- DNA polymerase — Enzyme that synthesizes DNA.
- Telomere — Non-coding DNA at chromosome ends.
- Telomerase — Enzyme that extends telomeres using RNA template.
Action Items / Next Steps
- Review the functions of all enzymes involved in replication.
- Understand the clinical significance of replication inhibitors.
- Study the telomere shortening hypothesis and the role of telomerase.