M03.02.03 Steps of DNA replication

DNA replication is a fundamental process in which a cell duplicates its DNA, allowing for genetic information to be passed to daughter cells during cell division. The following outlines the key steps and mechanisms involved in DNA replication, with a comparison between prokaryotic and eukaryotic systems.

Steps of DNA Replication

1. Recognition of the Origin of Replication
   • Prokaryotes: Single origin of replication (oriC).
   • Eukaryotes: Multiple origins of replication along the chromosome.
2. Unwinding of DNA
   • Helicase breaks hydrogen bonds between base pairs, creating two replication forks.
   • Single-stranded DNA binding proteins (SSB) stabilize the unwound DNA and prevent reassociation.
3. Synthesis of RNA Primers
   • Primase synthesizes a short RNA primer to initiate DNA synthesis.
4. DNA Synthesis
   • Leading Strand: Synthesized continuously in the 5′→3′ direction by DNA polymerase III (prokaryotes) or DNA polymerases α and δ (eukaryotes).
   • Lagging Strand: Synthesized discontinuously in short segments called Okazaki fragments. Each fragment starts with an RNA primer and is extended by DNA polymerase III (prokaryotes) or DNA polymerases α and δ (eukaryotes).
5. Removal of RNA Primers
   • Prokaryotes: DNA polymerase I removes RNA primers and fills in the gaps with DNA.
   • Eukaryotes: RNAase H removes RNA primers, and DNA polymerase δ fills in the gaps.
6. Joining of Okazaki Fragments
   • DNA ligase seals nicks between Okazaki fragments, forming a continuous DNA strand.
7. Relief of Supercoiling
   • DNA gyrase (Topoisomerase II) in prokaryotes and DNA topoisomerase II in eukaryotes relieve the positive supercoiling ahead of the replication fork by introducing negative supercoils.
8. Proofreading and Error Correction
   • Both prokaryotic and eukaryotic DNA polymerases have 3′→5′ Exonucleases activity to remove mispaired bases and ensure high-fidelity DNA replication.
9. Synthesis of Telomeres (Eukaryotes only)
   • Telomerase extends the telomeres at the ends of linear chromosomes to prevent the loss of genetic information.

Detailed Steps and Proteins Involved in DNA Replication

Leading Strand Synthesis: Continuous process where DNA polymerase extends the primer moving into the replication fork.

Lagging Strand Synthesis: Discontinuous process involving Okazaki fragments. Primase synthesizes primers, and DNA polymerase extends these fragments. RNase H (in eukaryotes) or DNA polymerase I (in prokaryotes) removes RNA primers, and DNA ligase joins fragments.

Notes on Telomerase and Reverse Transcriptase

• Telomerase: An enzyme in eukaryotes that maintains telomeres, preventing chromosome shortening. Active in embryonic, germ, and some stem cells, but not in most somatic cells. Elevated in many cancer cells, contributing to their immortality.
• Reverse Transcriptase: An RNA-dependent DNA polymerase found in retroviruses like HIV. Converts RNA into DNA. Eukaryotic cells also have reverse transcriptase activity associated with Telomerase and retrotransposons.

Pharmacological Connections

• Quinolones: Inhibit DNA gyrase, blocking bacterial DNA replication and transcription.
• Antiretroviral Drugs (e.g., AZT): Inhibit reverse transcriptase in retroviruses, preventing DNA synthesis from RNA templates.

References

1. Alberts, B., et al. (2014). Molecular Biology of the Cell. 6th ed. Garland Science.
2. Nelson, D. L., & Cox, M. M. (2017). Lehninger Principles of Biochemistry. 7th ed. W.H. Freeman.
3. Jorde, L. B., Carey, J. C., & Bamshad, M. J. (2019). Medical Genetics. 6th ed. Elsevier