lecture recording on 25 February 2025 at 16.29.07 PM

Overview of DNA Replication

• DNA replication is semi-discontinuous due to the antiparallel structure of DNA strands.

  • DNA is synthesized in the 5' to 3' direction.

  • The leading strand is synthesized continuously, while the lagging strand is synthesized in short fragments called Okazaki fragments.

Leading and Lagging Strands

• Leading Strand:

  • Synthesized continuously with a single RNA primer.

  • DNA polymerase can follow the fork moving in the same direction as the replication is occurring.

• Lagging Strand:

  • Synthesized in chunks (Okazaki fragments) due to its antiparallel nature.

  • Requires multiple RNA primers as DNA polymerase cannot synthesize in the opposite direction.

Okazaki Fragments and Polymerase Activity

• At the junction between Okazaki fragments, there are a 3' hydroxyl (OH) group and a 5' monophosphate, making it impossible for DNA polymerase to seal the gap.

Key Players in DNA Replication

• Helicase:

  • Unwinds the double helix at the replication fork to allow strand separation.

• Primase:

  • Synthesizes RNA primers that provide starting points for DNA synthesis.

• DNA Polymerase III (POL III):

  • Extends RNA primers and synthesizes new DNA strands.

  • Has proofreading capability; can reverse to fix mismatches by excising incorrect nucleotides using its 3' to 5' exonuclease activity.

• DNA Polymerase I (POL I):

  • Removes RNA primers from the lagging strand using its 5' to 3' exonuclease activity.

  • Replaces RNA with DNA, also capable of proofreading.

Removal of RNA Primers and Nick Sealing

• After RNA primers are removed:

  • DNA Polymerase I fills the gaps created.

  • The nicks in the DNA backbone are sealed by DNA ligase, completing the replication process.

Nucleases and Their Functions

• Nucleases: Broad term for enzymes that hydrolyze phosphodiester bonds in nucleic acids.

  • Classified as DNases (for DNA) and RNases (for RNA).

  • Can be broken down further into:

    • Endonucleases: Cleave bonds within a nucleic acid strand at specific sites.

    • Exonucleases: Remove nucleotides from the end of a nucleic acid chain.

Comparison of DNA Polymerases

• DNA Polymerase III:

  • Synthesizes DNA in 5' to 3' direction and has 3' to 5' proofreading capability.

• DNA Polymerase I:

  • Also synthesizes DNA in 5' to 3' direction but removes primers using 5' to 3' exonuclease activity.

Summary of Key Terms

• Exonuclease Activity: Ability of an enzyme to remove nucleotides from the ends of DNA/RNA.

• Proofreading: Mechanism used by DNA polymerases to correct mistakes during DNA synthesis.

• Nick Translation: Process of replacing RNA primers with DNA and sealing nicks in the DNA strand.