DNA Replication and Repair Practice Flashcards

Fundamentals of DNA Replication

• Base-Pairing Mechanism:

  • DNA replication is governed by specific base-pairing rules where Adenine ($A$) pairs only with Thymine ($T$), and Guanine ($G$) pairs only with Cytosine ($C$).
  • Each of the two strands in a DNA double helix can serve as a template. This template specifies the sequence of nucleotides for a new complementary strand.
  • The process is described as semiconservative, meaning each daughter DNA molecule consists of one original parental strand and one newly synthesized strand.

• Models of DNA Replication:

  • Semiconservative Model: The two parental strands separate, and each serves as a template for a new strand. Resulting DNA contains one old and one new strand.
  • Conservative Model: The entire parental double helix serves as a template for a whole new double helix; the original parental molecule is conserved intact.
  • Dispersive Model: Both strands of the daughter molecules contain a mixture of various fragments of parental and newly synthesized DNA.

• The Meselson-Stahl Experiment:

  • Methodology: Bacteria were grown for several generations in a medium containing the heavy nitrogen isotope ($^{15}N$) to label the DNA. They were then transferred to a light medium ($^{14}N$).
  • Observations:
    • After one generation in the light medium, the DNA produced a single band positioned midway between the heavy and light DNA densities. This result ruled out the conservative model.
    • After a second round of replication, two bands appeared: one midway and one at the light density. This ruled out the dispersive model.
  • Conclusion: The data definitively supported the semiconservative replication model.

Initiation and Origins of Replication

• Replication Origins:

  • DNA synthesis begins at specific sites called replication origins.
  • Prokaryotes (Bacteria): Typically contain a single origin of replication on a single circular chromosome.
  • Eukaryotes: Eukaryotic chromosomes are much larger and linear, requiring multiple replication origins to ensure the genome is copied in a timely manner.

• Replication Forks:

  • Two replication forks form at each origin. These forks move in opposite directions (bidirectionally) away from the origin as replication proceeds.
  • A "replication bubble" forms as the two forks move apart.

DNA Polymerases and the Synthesis Reaction

• Function of DNA Polymerase:

  • These enzymes synthesize new DNA using the parental strand as a template. They catalyze the covalent linkage of nucleoside triphosphates into the growing strand.
  • Eukaryotic Varieties: Eukaryotic cells contain at least five major DNA polymerases:
    • Polymerase $\alpha$, $\delta$, and $\epsilon$: Primarily involved in chromosomal DNA replication.
    • Polymerase $\gamma$: Responsible for the replication of mitochondrial DNA.
    • Polymerase $\beta$: Primarily functions in the repair of DNA damage.
  • Historical Figures: Arthur Kornberg and Sylvy Ruth Levy are credited with foundational work on DNA polymerase.

• Fundamental Properties of Polymerization:

  1. Nucleotides are only added to the $3'$ end of a growing DNA strand.
  2. DNA synthesis proceeds exclusively in the $5'\text{-to-}3'$ direction.

• Reaction Mechanism:

  • Nucleotides enter the reaction as high-energy deoxyribonucleoside triphosphates ($dNTPs$).
  • The incoming nucleoside triphosphate forms a base pair with its partner on the template strand.
  • The reaction is driven by the hydrolysis of a high-energy phosphate bond, releasing pyrophosphate ($PP_i$), which is subsequently hydrolyzed to inorganic phosphate ($P_i$) to make the reaction effectively irreversible.

The Asymmetrical Replication Fork

• Leading and Lagging Strands:

  • Because DNA polymerase only works in the $5'\text{-to-}3'$ direction, the replication fork is asymmetrical.
  • Leading Strand: Synthesized continuously toward the replication fork.
  • Lagging Strand: Synthesized discontinuously away from the replication fork as a series of short DNA fragments.

• Okazaki Fragments:

  • The short pieces of DNA synthesized on the lagging strand are called Okazaki fragments.
  • In eukaryotes, these fragments are typically about $200$ nucleotides long.

• RNA Primers:

  • DNA polymerase cannot start a new strand from scratch; it requires a primer.
  • Primase: An enzyme that synthesizes short lengths of RNA (primers) to provide the necessary $3'\text{-OH}$ group for DNA polymerase.
  • On the lagging strand, new primers are made at regular intervals.

• Joining the Fragments:

  1. Nucleases: Remove the RNA primers.
  2. Repair Polymerase ($\text{DNA Polymerase } \delta$ in eukaryotes): Fills the resulting gaps with DNA.
  3. DNA Ligase: Uses energy from ATP hydrolysis to join the fragments by linking the $5'$ phosphate of one fragment to the $3'$ hydroxyl of the next.

Polymerase Chain Reaction (PCR)

• Function: PCR is a laboratory technique used to amplify specific target DNA sequences, producing over a million-fold copies within a few hours.
• Taq Polymerase: The process utilizes a heat-resistant enzyme, Taq DNA polymerase, sourced from the bacterium Thermus aquaticus, which can tolerate the high temperatures required to denature the DNA double helix.

Challenges of DNA Replication

• Challenge 1: Torsional Stress:

  • The unwinding of the DNA double helix by DNA helicase (using ATP hydrolysis) creates torsional stress and supercoiling ahead of the replication fork.
  • Topoisomerases: Relieve this stress by generating temporary nicks in the DNA.
    • Topoisomerase I: Cleaves a single strand of DNA to allow rotation and unwinding.
    • Topoisomerase II: Cleaves both strands (double-strand break) to allow one double-stranded DNA segment to pass through another. It is a homodimer (Types IIA and IIB).
  • Two-Gate Mechanism of Type II Topoisomerase:
    1. G-segment (Gate segment) binding and bending; T-segment (Transport segment) capture at the N-gate.
    2. ATP binding traps the T-segment and triggers G-segment cleavage.
    3. The DNA-gate opens, pushing the T-segment through.
    4. G-segment is resealed; the C-gate opens to release the T-segment.
    5. Reset: ATP hydrolysis reopens the N-gate.
  • Specific Enzymes:
    • E. coli: Gyrase (Type IIA) and Decatenase (Type IV, for unlinking daughter chromosomes).
    • Human: Type $II\alpha$ (involved in cleavage, ligation, and contains Nuclear Localization Sequences/NLS).

• Challenge 2: Replication of Linear Chromosome Ends:

  • The "end-replication problem": DNA polymerase cannot complete the very $5'$ end of the lagging strand because there is no place for an RNA primer.
  • Telomerase: An enzyme that replicates the ends of eukaryotic chromosomes (telomeres). It extends the template strand of the lagging strand using its own built-in RNA template.
  • DNA Polymerase $\alpha$: Subsequently completes the lagging strand synthesis at the ends.

• Challenge 3: Replication Errors and DNA Damage:

  • Replication is highly accurate but not perfect, with an error rate of approximately one incorrect base per $10^9$ to $10^{10}$ nucleotides.

DNA Proofreading and Repair Mechanisms

• DNA Polymerase Proofreading:

  • The enzyme is self-correcting. It possesses two distinct sites: the Polymerizing mode (P) and the Proofreading/Editing mode (E).
  • If an incorrect nucleotide is added, the E site cleaves it from the strand, and the P site replaces it with the correct one.

• Spontaneous Chemical Damage:

  • Depurination: Spontaneous removal of Guanine or Adenine bases from DNA. Approximately $5000$ purine bases are lost per human cell per day.
  • Deamination: Converts Cytosine to Uracil (approximately $100$ bases per cell per day). If unrepaired, this leads to $CG > TA$ mutations.
  • Thymine Dimers: Caused by UV radiation in sunlight; two adjacent thymine bases become covalently attached. This is common in skin cells.

• Major Repair Pathways:

  1. Base Excision Repair (BER): Used to remove mismatched bases (e.g., Uracil). Apurinic endonuclease I (APE1) cuts the backbone at the abasic site. DNA Pol $\beta$ inserts the single base, and DNA ligase seals it.
  2. Nucleotide Excision Repair (NER): Recognizes large distortions in the double helix, such as thymine dimers. The XP (Xeroderma Pigmentosum) protein complex (XP-A through XP-G) repairs the damage. Mutations in these genes lead to Xeroderma Pigmentosum, increasing susceptibility to skin cancer (melanoma).
  3. Mismatch Excision Repair (MMR): Removes replication errors that escape proofreading. The MSH2-MSH6 complex binds the mismatch, an endonuclease removes the region, DNA Pol $\delta$ fills the gap, and ligase connects it.
     • Bacteria (E. coli) MMR: Uses MutS to recognize mismatches, MutL to form a complex, and MutH to cleave the unmethylated strand.
     • Clinical Link: Inheritable loss of MSH2 or MLH1 causes Lynch Syndrome (nonpolyposis colorectal cancer).

• Double-Strand Break Repair:

  • Nonhomologous End Joining (NHEJ): The broken ends are cleaned by a nuclease and joined. This often results in the loss of nucleotides at the repair site.
  • Homologous Recombination (HR): Uses the undamaged homologous double helix as a template for flawless repair.
  • Histone Involvement: Phosphorylation of the H2AX histone protein on Serine 139 directs these repair pathways.

Clinical Consequences of Mutation

• Sickle-Cell Anemia: A single nucleotide change in the $\beta$-globin gene causes a Glutamic Acid to Valine substitution at the sixth amino acid position. Two copies of this mutant gene result in the disease.
• Cancer and Aging:
  • Mutation incidence increases with age. Colon cancer results from the accumulation of multiple mutations.
  • Age-related CpG (arCpG) methylation: DNA methylation changes throughout life. Methylation in promoter regions can silence tumor suppressor genes, while methylation within the gene body can induce mutations.

Comparison of Bacterial and Eukaryotic Replication

Summary of Key DNA Replication Proteins