DNA Replication Lecture Notes

Introduction

• Purpose of DNA replication: Allows for cell replication, necessary for creating more cells.
• Occurs in the S phase of the cell cycle.
• Cell cycle: G1, S phase, G2, mitosis resulting in two identical daughter cells.

Key Concepts of DNA Replication

1. Semi-Conservative Model

• DNA replication is semi-conservative.
• Involves separating two parental (old) DNA strands.
• Each old strand serves as a template to synthesize a new complementary (daughter) strand.
  • Adenine pairs with Thymine, Guanine pairs with Cytosine.
• Results in two new double-stranded DNA molecules.

2. Direction of Replication

• DNA is replicated from the 5’ to 3’ direction.
  • 5’ end has a phosphate group, 3’ end has a hydroxyl group (OH).
• DNA strands are antiparallel.

3. Bidirectional Replication

• DNA replication is bi-directional, starting from the origin of replication.
• Creates replication forks where helicases unwind DNA.
• DNA polymerases synthesize new DNA.

Stages of DNA Replication

1. Initiation

• Origin of Replication:
  • Rich in adenine and thymine due to fewer hydrogen bonds.
  • Multiple origins in eukaryotic cells.
• Pre-Replication Protein Complex:
  • Binds to origin and separates the strands.
• Single-Stranded Binding Proteins:
  • Prevent re-annealing of strands, protect from nucleases.
• Helicase:
  • Unwinds DNA, requires ATP.

2. Elongation

• Primase:
  • Lays down RNA primers for DNA polymerase to start replication.
• DNA Polymerase III:
  • Synthesizes DNA, requires 3’ OH from RNA primer.
  • Works continuously on the leading strand towards the replication fork.
  • Works in fragments on the lagging strand (Okazaki fragments).
• Proofreading:
  • Checks for errors, uses exonuclease activity to correct mistakes.
• DNA Polymerase I:
  • Removes RNA primers, fills in gaps with DNA.
• Ligase:
  • Connects DNA fragments on the lagging strand.

3. Termination

• Occurs when replication forks meet.
• Replication stops when all DNA has been replicated.

Telomeres

• Telomeres shorten with each replication cycle, preventing gene loss.
• Do not code for RNA, sacrifice themselves to protect genes.
• Telomerase:
  • Enzyme that elongates telomeres using reverse transcription.
  • Important in stem cells and cancer cells.

Clinical Application

• Topoisomerases:
  • Alleviate supercoiling during replication.
  • Targeted by drugs in cancer therapy (e.g., Etoposide) and antibiotics (e.g., Fluoroquinolones).
• HIV Therapy:
  • Nucleoside Reverse Transcriptase Inhibitors (NRTIs) inhibit replication in HIV-infected cells by blocking DNA polymerase activity.

Conclusion

• DNA replication is essential for cell division.
• Involves complex enzymatic processes ensuring genetic fidelity.
• Clinical relevance in understanding disease mechanisms and drug targets.