02-04-2025 Lecture Outline 1

Replication

DNA Replication Overview

• DNA double-helix unwinds in the region to be replicated.

• Each DNA strand serves as a template for the synthesis of a new strand.

• The site where strands unwind is known as the Replication Fork.

DNA Replication Mechanism

• Individual deoxyribonucleoside triphosphates (nucleotides) are added sequentially to the growing end of each new strand.

• The order of base addition is determined by pairing incoming nucleotide bases to template strand bases.

• The outcome is two new semiconservative double strands.

Role of DNA Helicase

• DNA strands must be separated and unwound before replication; the stability of the double helix makes separation unfavorable.

• DNA Helicase binds and hydrolyzes ATP molecules, undergoing conformational changes that allow it to unscrew and pry apart the two strands as it progresses along the DNA.

Single-Strand Binding Proteins (SSBPs)

• Bind to open single-strand regions of DNA.

• Prevent self-pairing or re-annealing while keeping bases accessible for replication.

DNA Primase

• Synthesizes an initial “primer” strand (10 to 200 nucleotides) onto the template strand.

• The primer strand is essential for initiating replication, constructed from rNTPs, resulting in an RNA primer attached to the DNA template.

Leading Strand Synthesis

• A new DNA strand is synthesized only in the 5’ to 3’ direction.

• For the template strand oriented 3’ to 5’, this results in continuous addition of nucleotides.

• This strand is referred to as the Leading Strand.

Lagging Strand Synthesis

• The complementary template strand is oriented 5’ to 3’, which cannot be synthesized continuously.

• Requires synthesis in the 3’ to 5’ direction, which is not feasible.

• This strand is synthesized in pieces known as Okazaki Fragments.

Okazaki Fragments

• Lagging strand synthesis results in short, unconnected sequences.

• These fragments must be ligated together by the enzyme DNA Ligase as replication proceeds.

The Need for RNA Primer

• Initial primer synthesis is challenging and prone to errors.

• Using RNA allows for easy replacement with DNA, providing an opportunity to correct mistakes.

• Initial RNA is assumed to have errors, which are corrected during replacement.

Role of Topoisomerase

• Topoisomerase I: Relieves accumulated strain from supercoiling by making single-strand cuts in the DNA.

• Topoisomerase II: Introduces double-strand breaks, allowing one helix to pass through another before resealing the break, thus managing supercoiling during replication.

Replication Machine Components

• The replication machinery consists of:

  • DNA Helicase: Unwinds DNA.

  • Primase: Synthesizes RNA primers.

  • Polymerase: Adds new nucleotides to the growing strands.

  • Single-Strand Binding Proteins: Stabilize unwound DNA strands.

  • Clamp Loader and Sliding Clamp: Ensure DNA polymerase remains in place during synthesis.

Maintenance of DNA Sequences

• Mutations, or changes in DNA sequences, result from failures in repair mechanisms.

• A mutation rate is approximately 1 base per 10^9 nucleotides per replication, and most mutations are silent (not affecting the phenotype).

Proofreading Mechanisms

• The rate of replication mistakes significantly exceeds the mutation rate.

• Primary mechanisms include:

  • DNA Polymerase Proofreading: Correct base pairing leads to conformational change and bond formation, while incorrect base pairing leads to stalling and dissociation of the incorrect base.

  • Exonucleolytic Proofreading: Wrong bases are removed by the nuclease activity of the polymerase when tautomeric forms of bases are bonded, reverting to correct base pairing.

Telomeres and Telomerase

• Telomeres prevent DNA molecules from becoming shorter with each replication, avoiding issues at the ends of chromosomes.

• Telomerase: A ribonucleoprotein complex that extends the telomeres by utilizing an RNA template for elongation in the 5’ to 3’ direction, allowing DNA polymerase to bind.

Telomere Length Regulation

• Regulated by cells; synthesis of telomerase can be turned off or slowed down with time or defects in chromosomes.

• Insufficient telomerase can lead to cell death or replicative senescence, contributing to certain cancers.