9_Replication_Sp2025_abb

Learning Objectives

• Replication Objectives:

• Illustrate and compare the three models proposed for DNA replication:

  • Semiconservative

  • Conservative

  • Dispersive

• Describe and interpret experimental evidence supporting the semiconservative nature of DNA replication.

• Predict mechanisms of replication based on results from experiments like Meselson and Stahl.

• Differentiate between theta vs. linear replication.

• Summarize the stages of replication:

  • Initiation

  • Unwinding

  • Elongation

  • Termination

• Identify and describe the roles of key proteins involved in replication.

• Predict replication impacts from the absence of specific replication proteins.

• Sketch a replication bubble showing various components (polarity of strands, leading vs lagging strands, Okazaki fragments, RNA primers).

• Explain the end replication problem and how telomeres and telomerase address this issue in linear chromosomes.

• Describe amplifying DNA fragments through PCR and compare this process with DNA replication.

• Given DNA sequences, provide sequences for two PCR amplification primers.

• Predict PCR product outcomes based on primer and template sequences.

• Discuss limitations of PCR.

DNA Replication Accuracy

• Speed of Replication:

  • E. coli: ~1000 nucleotides/second.

  • Humans: ~50 nucleotides/second.

• High Accuracy:

  • Only 1 mistake per 10 million nucleotides.

• Mechanisms Ensuring Accuracy:

  • DNA Polymerase Efficiency: Occurrence of mismatches is low (10^-5).

  • Proofreading Mechanism: 3' → 5' exonuclease activity.

  • DNA Repair Mechanisms: As described in later chapters.

• Errors are common during copying, increasing the possibility with more replication cycles.

Historical Context

• Key Date: April 25, 1953

• Discussion of DNA's molecular structure and implications for genetic material copying methods.

Proposed Mechanisms for DNA Replication

• Semiconservative Replication:

  • Each daughter strand comprises one parental and one newly synthesized strand.

• Conservative Replication:

  • One daughter duplex contains both parental strands; the other contains both daughter strands.

• Dispersive Replication:

  • Daughter duplexes contain mixed parental and daughter segments.

Meselson and Stahl Experiment

• Methodology:

  • E. coli grown in 15N medium, transferred to 14N medium.

  • DNA separation via equilibrium density gradient ultracentrifugation using CsCl.

• Expected Results:

  • Bands of varying densities indicate the mechanism of replication:

    • Semiconservative model confirmed via density gradient outcomes.

Stages of DNA Replication in E. coli

• Initiation: Begins at OriC (245 bp, A-T rich).

• Unwinding: Key proteins include helicase, SSBs, DNA gyrase.

• Elongation:

  • Primase synthesizes RNA primers.

  • DNA polymerase III continues synthesis in the 5' → 3' direction.

  • Lagging strand synthesis via Okazaki fragments.

  • DNA polymerase I and ligase finalize replication by removing RNA primers and connecting fragments.

• Termination: Completion of replication at specific sites.

End-Replication Problem in Eukaryotes

• Telomeres:

  • Repetitive DNA sequences at chromosome ends preventing loss of important genetic information.

• Telomerase: Alleviates the end replication problem by adding repeats.

  • Consists of TERC (RNA component) and TERT (reverse transcriptase).

  • T-loop formation protects chromosome ends.

PCR (Polymerase Chain Reaction)

• Amplification of specific DNA sequences involving:

  • Short synthetic single-stranded DNAs as primers.

• Requirements:

  • dsDNA template, dNTPs, two primers, buffer solution, thermocycler, and thermo-stable polymerase.

• Process:

  • Three cycles: Denaturation, primer annealing, and extension.

  • DNA doubles with each cycle.

Applications and Limitations of PCR

• Uses:

  • Amplification for cloning, diagnostics, forensic analysis, etc.

• Limitations:

  • Requires prior knowledge of DNA sequences; susceptible to contamination; lacks proofreading activity in Taq polymerase.