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CELS191 Lecture 13: DNA Replication

Overview of DNA Replication

  • DNA or RNA synthesis always occurs in the 5’ → 3’ direction.
  • Parental DNA template strands move in the 3’ → 5’ direction.

Objectives of Lecture 13

  • Describe the mechanism of DNA replication and the specific functions of all necessary molecules.
  • Explain how errors in the DNA sequence can be corrected and why this is important.

Key Mechanisms of DNA Replication

  • Semi-discontinuous synthesis

    • Leading strand: synthesized continuously in the 5’ → 3’ direction.
    • Lagging strand: synthesized discontinuously in fragments called Okazaki fragments.

  • Replication Fork: Area where the DNA strands separate for replication.

  • Replication Bubbles: Formed when multiple origins of replication in eukaryotic chromosomes open up.

Directions of Synthesis

  • New DNA strands are formed by progressively adding nucleotides (A, C, T, G).
  • The unwinding of double-stranded DNA by helicase reveals parental templates.
  • Topoisomerase helps relieve torsional strain during unwinding.
  • Single-stranded DNA binding proteins prevent the strands from re-annealing.

Enzymes Involved in DNA Replication

  1. Primase
    • Synthesizes an RNA primer providing a starting point for nucleotide addition.
  2. DNA Polymerase III (Pol III)
    • Main enzyme for adding nucleotides to the growing DNA strand; requires an OH group.
    • Error rate: 1 in 10^8 - 10^10 base pairs.
    • Has a proofreading mechanism utilizing 3’ to 5’ exonuclease activity to remove incorrect nucleotides.
  3. DNA Polymerase I
    • Removes RNA primers (RNase H activity) and fills the gap with DNA nucleotides.
  4. DNA Ligase
    • Joins Okazaki fragments by forming phosphodiester bonds after RNA primers are replaced.

Repair of DNA Errors

  • During replication: Errors corrected using exonucleases (e.g., DNA Pol III).
  • After replication: Errors corrected using endonucleases; can be caused by environmental factors (e.g., radiation, chemical changes).
  • Importance of correcting errors: Uncorrected errors may lead to permanent mutations in the DNA sequence.

DNA Error Correction Mechanisms

  • 3’ to 5’ Exonuclease Activity:
    • Removes any incorrectly inserted bases during DNA synthesis.
  • Endonuclease Activity:
    • Repairs incorrectly inserted bases after replication is complete, involving cutting the DNA strand and synthesizing new DNA.

Importance of Correcting DNA Errors

  • Unchecked errors can lead to mutations, thereby altering future DNA replication and gene expression.
  • Correct base pairing is crucial; incorrect bases can lead to lasting changes in the genome, such as changes from C-G to A-T.

Summary of Key Concepts

  • DNA replication is semidiscontinuous: one strand synthesized continuously (leading strand) and one in fragments (lagging strand).
  • Multiple replication bubbles exist in eukaryotic cells to ensure efficiency and accuracy.
  • Key enzymes (polymers and ligases) play critical roles in both synthesis and error correction processes.