DNA and the Gene: Synthesis and Repair

Learning Objectives in DNA Synthesis and Repair

  • Interpretation of DNA Replication

    • Semiconservative Replication: Based on the Meselson-Stahl experiment, students should argue that DNA is replicated in a semiconservative manner.

  • Sequence Prediction: Students should be able to predict the sequences and polarities of synthesized DNA molecules based on the given template DNA.

  • Enzymes Involved:

    • Leading Strand: Understand the specific enzymes responsible for leading strand replication

    • Lagging Strand: Identify the enzymes involved in lagging strand replication and articulate their roles.

  • End Replication Problem: Explain why the 3' end of a chromosome cannot be replicated. Discuss how telomerase addresses this issue.

  • DNA Repair Mechanisms: Describe the processes of proofreading, mismatch repair, and nucleotide excision repair and their significance in maintaining DNA integrity.

Structure of DNA

  • Basic Structure:

    • Sugar-Phosphate Backbone: Composed of deoxyribonucleotides connected by phosphodiester linkages.

    • Antiparallel Strands: The strands have 5'-3' polarities running in opposite directions:

    • 5' End: The end with a phosphate group.

    • 3' End: The end with a hydroxyl group.

  • Complementary Base Pairing:

    • Hydrogen Bonds: Bases project from the sugar-phosphate backbone and pair through hydrogen bonds. The pairs are:

    • A with T

    • G with C

DNA Replication

Purpose of DNA Replication

  • To ensure that each new cell receives an exact copy of the DNA upon cell division.

Semiconservative Replication Model

  • Use a diagram to illustrate DNA before and after replication (first and second rounds).

Starting DNA Replication

  • Origin of Replication: A specific sequence where replication begins and replication bubbles form with two replication forks.

  • DNA Polymerase:

    • Enzyme responsible for synthesizing DNA

    • Adds deoxyribonucleotides to the 3' end, functioning in a 5' → 3' direction.

    • Cannot initiate synthesis de novo; requires a nucleotide to provide a 3' end.

    • Uses deoxyribonucleoside triphosphates (dNTPs) as building blocks.

Synthesis of Leading Strand

  • Steps involved:

    1. Opening and Unwinding:

    • Topoisomerase relieves twisting forces on DNA.

    • Helicase unwinds the double helix.

    • Single-strand DNA-binding proteins (SSBPs) stabilize the unwound strands.

    1. Priming:

    • Primase synthesizes an RNA primer that pairs with the DNA template.

    1. Extension:

    • DNA polymerase synthesizes the leading strand toward the replication fork, in a continuous manner.

Synthesis of Lagging Strand

  • Characteristics:

    • It is synthesized away from the replication fork in a discontinuous manner.

  • Okazaki Fragments: Short DNA segments synthesized during lagging strand replication.

    • Steps involved in synthesis:

    1. Primase synthesizes RNA primer.

    2. DNA polymerase III synthesizes the Okazaki fragments in a 5' → 3' direction.

    3. Each primer is replaced by DNA polymerase I, which removes the RNA and fills in with DNA.

    4. DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond.

Problems at Chromosome Ends

  • DNA Replication Challenges:

    • At the end of a chromosome, the lagging strand cannot be fully replicated due to the lack of an available primer, leading to unreplicated ends.

    • Consequence: Chromosomes may shorten by 50 to 100 nucleotides with each replication cycle.

  • Telomeres:

    • Non-coding, repetitive DNA sequences at chromosome ends

    • Prevent degradation and loss of essential genes.

Telomerase Function

  • Telomerase Action:

    • An enzyme that extends telomeres, especially in germ cells, to prevent loss during replication.

    • Somatic cells generally lack telomerase, resulting in gradual shortening with age.

    • Most cancer cells show active telomerase, allowing for unrestricted cell division.

Telomere Replication Process

  1. Unreplicated End: A parental strand remains after primer removal from the lagging strand.

  2. Telomerase Extends: It binds to the 3' end, using its RNA template to extend the strand.

  3. Repetition: Telomerase shifts along the strand, continuing to add repeats.

  4. Synthesis Completion: Standard DNA synthesis occurs to convert the extended single-stranded DNA into double-stranded DNA.

DNA Error Correction Mechanisms

  • Proofreading by DNA Polymerase:

    • The enzyme can identify and remove mismatched bases through its epsilon (ε) subunit, improving error rates significantly:

    • From 1 in 100,000 to about 1 in 10 million base pairs.

  • Mismatch Repair: Specific enzymes correct any mismatches remaining after synthesis.

  • Overall Error Rate: Approximately 1 mistake per billion bases.

Nucleotide Excision Repair

  • DNA Damage Causes: Harmful agents such as sunlight, X-rays, and chemicals can cause DNA lesions, e.g., thymine dimers from UV light.

  • Impact on Replication: These dimers introduce a kink, which obstructs the normal replication process, necessitating repair.