Overview
This lecture covers the core mechanisms and steps of DNA replication, including its purpose, process, key enzymes, and clinical applications.
Purpose and Fundamentals of DNA Replication
- DNA replication enables cells to produce identical genetic material for cell division.
- Occurs during the S phase of the cell cycle.
- Each chromosome is duplicated to produce two identical daughter cells.
- DNA replication follows the semi-conservative model: each new DNA has one old strand and one new strand.
- Synthesis proceeds in the 5’ to 3’ direction and is bi-directional from multiple origins.
Stages of DNA Replication
- Three main stages: initiation, elongation, and termination.
- Initiation starts at origins of replication, often rich in adenine (A) and thymine (T) due to easier bond breaking.
- Single-stranded binding proteins stabilize separated parental strands and prevent re-annealing or nuclease digestion.
- Helicase unwinds the DNA at replication forks; topoisomerases relieve DNA supercoiling ahead of replication forks.
Key Enzymes and Processes
- Primase synthesizes short RNA primers to provide a 3’ OH group for DNA polymerase.
- DNA polymerase III synthesizes DNA in the 5’→3’ direction and proofreads with 3’→5’ exonuclease activity.
- Leading strand: synthesized continuously; lagging strand: synthesized discontinuously as Okazaki fragments (RNA primers + DNA).
- DNA polymerase I removes RNA primers with 5’→3’ exonuclease activity and fills gaps with DNA; also proofreads.
- DNA ligase seals nicks between Okazaki fragments on the lagging strand.
Clinical Applications
- Topoisomerase inhibitors (e.g., irinotecan, etoposide for cancer; fluoroquinolones for bacterial infections) block DNA replication in target cells.
- Nucleoside reverse transcriptase inhibitors (NRTIs) for HIV block DNA synthesis by lacking the 3’ OH group required for elongation.
Termination and Telomeres
- Replication ends when polymerases meet at converging replication forks.
- Telomeres are non-coding ends of chromosomes that shorten with each replication cycle, preventing gene loss.
- Telomerase extends telomeres in stem and cancer cells using reverse transcription, allowing continuing replication.
- The Hayflick limit is the maximal number of divisions before telomere shortening threatens genes.
Key Terms & Definitions
- Semi-conservative replication — Each new DNA molecule contains one old and one new strand.
- Replication fork — Y-shaped region where DNA is unwound for replication.
- Okazaki fragments — Short DNA fragments made on the lagging strand.
- Topoisomerase — Enzyme relieving DNA supercoiling during replication.
- Telomere — Non-coding end region of chromosomes.
- Telomerase — Enzyme that extends telomeres using reverse transcriptase activity.
- Hayflick limit — Maximum cell divisions before critical telomere shortening.
Action Items / Next Steps
- Review enzyme functions in DNA replication.
- Memorize the order and roles of replication steps and main enzymes.
- Understand clinical relevance of replication inhibitors for cancer, bacterial infections, and HIV.