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.

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