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.