DNA Replication and Repair Flashcards

Fundamentals of DNA Replication

• Base-pairing as the Basis of Replication: DNA replication is fundamentally driven by the specific complementary base-paring of nucleotides.

  • Adenine ($A$) always pairs only with Thymine ($T$).

  • Guanine ($G$) always pairs only with Cytosine ($C$).

• Template Mechanism: Both strands of the parent DNA double helix serve as a template. Each strand specifies the sequence of nucleotides in its new complementary partner strand.

• Semiconservative Nature: DNA replication is "semiconservative," meaning each new DNA double helix consists of one strand from the original parental molecule and one newly synthesized strand.

Experimental Evidence for Replication Models

• The Meselson–Stahl Experiment: This experiment was crucial in determining the accurate model of DNA replication. Bacteria were grown for several generations in a medium containing either weight isotopes ($15N$, heavy) or ($14N$, light) to label the nitrogenous bases of their DNA.

• Rejection of the Conservative Model: When bacteria grown in the heavy medium ($15N$) were transferred to a light medium ($14N$), they produced a band of DNA that was positioned midway between the heavy and light positions after one round of replication. This provided evidence against the conservative model, which would have produced two distinct bands (one fully heavy, one fully light).

• Rejection of the Dispersive Model: After a second round of replication, the dispersive model was ruled out because it would have produced a single hybrid band that gradually shifted toward the light position. Instead, the results supported the semiconservative model, showing both a hybrid band and a light band.

. Initiation and Origins of Replication

• Replication Origins: DNA synthesis begins at specific sites called replication origins.

• Replication Forks: At each replication origin, two replication forks form. These forks move in opposite directions (away from each other) as replication proceeds.

• Eukaryotic Chromosomes: Unlike bacteria, which typically have a single circular chromosome with one origin, eukaryotic chromosomes are linear and contain multiple replication origins to manage the duplication of much larger genomes.

• Replication Bubbles: The area where the parental DNA strands have separated and new DNA has been synthesized is known as a replication bubble.

DNA Polymerases and the Mechanism of DNA Synthesis

• Basic Function: DNA polymerase is the enzyme that synthesizes new DNA using the parental strand as a template.

• Eukaryotic DNA Polymerases: There are five primary types of DNA polymerases in eukaryotic cells:

  • Polymerase $\alpha$: Involved in chromosomal DNA replication, specifically in the initiation of fragments.

  • Polymerase $\delta$: Involved in chromosomal DNA replication and filling gaps left by primers.

  • Polymerase $\epsilon$: 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 Attribution: Significant work on DNA polymerase was conducted by Arthur Kornberg and Sylvy Ruth Levy.

• Chemical Properties of Synthesis:

  1. Directionality: DNA polymerase only adds nucleotides to the $3'$ end of a growing strand. Synthesis always occurs in the $5'$ to $3'$ direction.

  2. Substrates: Nucleotides enter the reaction as deoxyribonucleoside triphosphates ($dNTPs$).

  3. Energetics: The incoming nucleoside triphosphate forms a base pair with the template. The reaction is driven by the hydrolysis of a high-energy phosphate bond, resulting in the release of pyrophosphate ($PP_i$).

The Asymmetrical Replication Fork: Leading and Lagging Strands

• Fork Asymmetry: Because DNA polymerase can only synthesize in the $5'$-to-$3'$ direction, the two strands at the replication fork are produced differently.

• Leading Strand: This strand is synthesized continuously in the $5'$ to $3'$ direction toward the replication fork.

• Lagging Strand: This strand is synthesized discontinuously in the direction away from the fork. It is made as a series of short fragments called Okazaki fragments, which are also synthesized in the $5'$ to $3'$ direction.

• Coordination: Both strands are synthesized simultaneously by a complex replication machine.

RNA Primers and the Coordination of Lagging Strand Synthesis

• Requirement for Primers: DNA polymerase cannot start a new strand from scratch; it requires a short length of RNA known as a primer provided by the enzyme primase.

• Eukaryotic Primer Spacing: In eukaryotes, RNA primers are placed at intervals of approximately $200$ nucleotides along the lagging strand template.

• Processing of Fragments:

  1. Nucleases: These enzymes remove the RNA primers.

  2. DNA Polymerase $\delta$: Fills the resulting gaps with DNA nucleotides.

  3. DNA Ligase: Joins the fragments together. This enzyme uses a molecule of $ATP$ to activate the $5'$ phosphate of one fragment before linking it to the $3'$ hydroxyl of the next.

Polymerase Chain Reaction (PCR)

• Capability: PCR can produce over a million-fold copies of a target DNA sequence within a few hours.

• Source of Enzymes: The heat-resistant enzyme used is Taq DNA polymerase, which is sourced from the bacterium Thermus aquaticus. This organism can tolerate high temperatures, allowing the enzyme to survive the denaturation steps of the PCR cycle.

Torsional Stress and the Role of Topoisomerases

• The Challenge of Stress: As DNA unwinds during replication and transcription, torsional stress develops in eukaryotic, bacterial, and mitochondrial genomes.

• Relief Mechanisms:

  • Supercoiling: Additional coiling of the double helix to form supercoils.

  • Topoisomerases: Enzymes that relieve stress by creating temporary nicks in the DNA to allow rotation around single strands.

• Types of Topoisomerases:

  • Topoisomerase I: Found in all cells; it cleaves one strand of the DNA to allow unwinding, producing a relaxed-circle conformation.

  • Topoisomerase II: Required to relieve greater tension than Topoisomerase I. It acts as a homodimer (types IIA and IIB in eukaryotes).

  • Bacterial Topoisomerases: Includes Gyrase (Type IIA) and Decatenase (Type IV), the latter of which unlinks daughter chromosomes.

  • Human Topoisomerase II $\alpha$: Contains an active site tyrosyl residue that binds DNA and a C-terminal domain with nuclear localization sequences ($NLS$).

• The "Two-Gate" Mechanism of Type II Topoisomerase:

  1. Binding: G-segment binding and bending occurs; the N-gate captures the T-segment.

  2. Cleavage: Binding of two $ATP$ molecules traps the T-segment and cleaves the G-segment.

  3. Transport: The DNA-gate opens, and the T-segment is pushed through.

  4. Release: The G-segment is resealed, and the C-terminal (C-gate) opens to release the T-segment.

  5. Reset: $ATP$ hydrolysis causes the N-gate to reopen for the next cycle.

Challenges of Linear Chromosome Ends: Telomeres and Telomerase

• The End-Replication Problem: DNA polymerase cannot complete the ends of the lagging strand of linear chromosomes because there is no place to put an RNA primer.

• Telomerase: This enzyme replicates the ends of eukaryotic chromosomes. It carries its own built-in RNA template to extend the template strand ($3'$ end) of the parental DNA.

• Completion: Once the template is extended, DNA polymerase $\alpha$ can copy the ends using its own primer, ensuring chromosomal length is maintained.

DNA Damage: Spontaneous and Environmental Causes

• Spontaneous Reactions:

  • Depurination: The removal of guanine or adenine from DNA. Each human cell loses approximately $5000$ purine bases ($A$ and $G$) daily.

  • Deamination: The conversion of cytosine to uracil occurs at a rate of about $100$ bases per cell per day. If unrepaired, this leads to $CG$ to $TA$ mutations.

• Environmental Damage:

  • Ultraviolet (UV) Radiation: Causes the covalent attachment of adjacent thymine bases, forming thymine dimers. Skin cells are particularly susceptible to this.

DNA Proofreading and Excision Repair Mechanisms

• DNA Polymerase Proofreading: DNA polymerase is self-correcting. It possesses two distinct modes:

  • Polymerizing Mode ($P$..

  • Editing Mode ($E$): The enzyme proofreads; if an incorrect base is detected, the $E$ site cleaves it from the strand, allowing the $P$ site to replace it correctly.

• Accuracy: Proofreading ensures that only one incorrect base is incorporated per $10^9$ to $10^{10}$ nucleotides.

• Base Excision Repair: Removes mismatched bases such as $T$ and replaces them with $C$. Apurinic endonuclease I (APE1) cuts the backbone, DNA Pol $\beta$ inserts the single base, and ligase seals it.

• Nucleotide Excision Repair: Recognizes double-helix distortions like thymine dimers. The XP protein complex (Xeroderma Pigmentosum proteins $XP-A$ through $XP-G$) handles this repair. Mutations in these genes predispose individuals to UV-induced skin cancers.

• Mismatch Excision Repair: Removes replication errors that escaped proofreading. The MSH2-MSH6 protein complex binds to the mispaired segment. An endonuclease removes the nucleotides, DNA Pol $\delta$ fills the gap, and ligase connects it.

• Bacterial Methyl Mismatch Repair (MMR): Uses MutS to recognize the mismatch, MutL to form a complex, and MutH to cleave the unmethylated $GATC$ sequence.

Repair of Double-Stranded DNA Breaks

• Nonhomologous End Joining: The break is "cleaned" by a nuclease that chews back the ends to create flush ends. These ends are then joined. This process often results in the loss of nucleotides at the repair site.

• Homologous Recombination: Uses an undamaged double helix (sister chromatid) as a template to flawlessly repair the damaged double helix, resulting in no loss of information.

• Histone Involvement: The phosphorylation of the H2AX histone protein on the serine 139 residue directs the repair pathway toward either homologous recombination or nonhomologous end-joining.

Clinical and Physiological Implications of DNA Mutation

• Sickle-Cell Anemia: Caused by a single nucleotide change that results in a glutamic acid to valine substitution at the sixth amino acid position in $\beta$-globin. Possessing two copies of this mutant gene causes the disease.

• Lynch Syndrome (Nonpolyposis Colorectal Cancer): Caused by inheritable loss-of-function mutations in MSH2 or MLH1 proteins involved in mismatch repair.

• Xeroderma Pigmentosum: Deficiency in nucleotide excision repair leading to high susceptibility to melanoma and squamous cell carcinoma.

• Cancer and Aging: The incidence of cancer (e.g., colon cancer) increases with age due to the accumulation of multiple mutations and age-related changes in DNA methylation.

  • Age-Related CpG (arCpG): Studies of $1006$ whole blood samples show methylation levels change with age.

  • Methylation Effects: Methylation in the promoter regions of tumor suppressor genes can silence them, while methylation within the gene body can induce mutational events.

Comparison: Bacterial vs. Eukaryotic DNA Replication

Summary of Proteins Involved in DNA Replication

• DNA Polymerase: Catalyzes the addition of nucleotides to the $3'$ end.

• DNA Helicase: Uses $ATP$ hydrolysis to unwind the double helix.

• Single-strand DNA-binding protein: Prevents base pairs from re-forming on the lagging strand template.

• DNA Topoisomerase: Produces transient nicks to relieve tension ahead of the helicase.

• Sliding Clamp: Keeps DNA polymerase attached to the template strand.

• Clamp Loader: Uses $ATP$ hydrolysis to lock the sliding clamp onto DNA.

• Primase: Synthesizes RNA primers on the lagging strand.

• DNA Ligase: Joins Okazaki fragments using $ATP$ hydrolysis.