DNA Replication & Repair

DNA Replication & Repair

• DNA replication is a process that creates a new double-stranded DNA molecule. Replication begins with the separation of the two strands of double-stranded DNA.
• Learning objectives include understanding DNA replication processes, the molecular details, and the importance of DNA repair and mutations.
• DNA is reproduced by semiconservative replication.
• The complementarity of DNA strands allows each strand to act as a template for the other during synthesis.
• The Meselson-Stahl experiment demonstrated semiconservative replication, showing that each new DNA molecule consists of one original strand and one new strand.

Three Models of DNA Replication

• Conservative: Original DNA molecule remains intact, and a completely new molecule is created.
• Semiconservative: Each new molecule contains one original and one new strand.
• Dispersive: DNA molecules are composed of fragments of both original and new DNA.

The Meselson-Stahl Experiment

• The experiment used isotopes of nitrogen (15N and 14N) to label DNA.
• Bacteria were initially cultured in a nutrient-rich medium containing a heavier isotope of nitrogen, 15N, which allowed for the incorporation of this isotope into newly synthesized DNA strands. After sufficient growth, the bacteria were then transferred to a lighter nitrogen medium, 14N. DNA samples were extracted at various intervals to monitor the progression of replication.
• Centrifugation of DNA samples involved spinning the samples at high speeds, causing denser molecules to move toward the bottom of the tube while lighter ones remained at the top. This technique enabled the researchers to observe distinct bands corresponding to the different densities of DNA, thereby providing critical evidence for validating the proposed models of DNA replication, including semiconservative and dispersive mechanisms.

Where Replication Occurs in the Cell Cycle

• DNA replication occurs during the S phase of interphase.
• Interphase includes G1, S, and G2 phases.
• During S phase, each chromosome is duplicated by the cell.
• G2 is when the cell checks the duplicated chromosomes for errors.

Origins of Replication

• Replication begins at specific sites on the DNA called origins of replication.
• In prokaryotes (bacteria), replication typically has one origin of replication.
• In eukaryotes (e.g., human cells), there are multiple origins of replication.
  • This allows replication to proceed faster and is associated with a replication bubble and replication forks.
  • replication forks are where the DNA strands separate to allow replication.

Four Stages of Replication

• 1. Initiation: Topoisomerase relieves strain ahead of the replication fork, and helicase unwinds the DNA double helix. Single-strand binding proteins prevent the strands from re-annealing.
• 2. Unwinding: Helicase breaks hydrogen bonds, separating the DNA strands.
• 3. Primer Synthesis: Primase synthesizes RNA primers to provide a 3' end for DNA polymerase.
• 4. Elongation: DNA polymerase extends the RNA primers with DNA nucleotides.

Nucleotide Incorporation

• Nucleotides are added to the 3' end of the growing strand.
• Two phosphate groups are lost as they join, providing energy for the reaction.
• DNA polymerase adds complementary nucleotides to the template strand.

Bacterial DNA Replication: Leading and Lagging Strands

• In bacterial DNA, replication proceeds in both directions from a single origin.
• The leading strand is synthesized continuously towards the replication fork, allowing for efficient elongation.
• The lagging strand is synthesized discontinuously in short segments known as Okazaki fragments, which are later joined together by DNA ligase to form a continuous strand.
• Okazaki fragments are short fragments of DNA synthesized on the lagging strand.
• DNA ligase joins the Okazaki fragments together.

DNA Repair Mechanisms

• DNA replication is a complex process that is not perfectly accurate. During this essential cellular function, initial mistakes in nucleotide incorporation can occur naturally, leading to mutations if not corrected. These errors can arise from various factors, including the inherent limitations of the DNA polymerase enzyme that is responsible for synthesizing the new DNA strand.
• DNA polymerase can make mistakes in adding the right nucleotides, so proofreading is needed. This proofreading process involves DNA polymerase participating in a secondary function where it can detect and excise incorrectly paired nucleotides. This action greatly enhances the fidelity of DNA replication, reducing the mutation rate effectively.
• DNA repair mechanisms ensure accuracy, thus maintaining genomic stability. These mechanisms are critical for cellular health and prevent the accumulation of mutations that can lead to diseases, including cancer. Several sophisticated pathways actively monitor and repair damaged DNA throughout the cell cycle.
• MMR (Mismatch Repair) corrects errors in base pairing during replication, which is crucial in maintaining genetic integrity. This mechanism recognizes mispaired bases that escape proofreading, removes the erroneous segment of DNA, and fills in the correct nucleotides using the complementary strand as a template, thus restoring the sequence.

DNA Damage and Repair

• Various damaging agents (e.g., X-rays, UV, chemicals) can cause damage to DNA. These agents can lead to different forms of DNA damage, ranging from single-strand breaks to more complex lesions that may disrupt the DNA structure.
• Several repair processes exist to fix the breaks and abnormalities in the DNA molecule. Depending on the type of damage, different pathways are employed, such as Base Excision Repair (BER) for small, non-helix-distorting base lesions, or Homologous Recombination (HR) that is utilized for repairing double-strand breaks, ensuring that the integrity of the genetic material is preserved.
• Base Excision Repair (BER) is a critical cellular mechanism responsible for correcting small, non-helix-distorting base lesions in DNA. This process primarily targets damages caused by oxidative stress, alkylation, and spontaneous deamination, which can inevitably lead to mutations if left uncorrected. The BER pathway is initiated when a specific DNA glycosylase recognizes and removes the damaged base, creating an apurinic or apyrimidinic (AP) site. Subsequently, an AP endonuclease cleaves the DNA backbone at the AP site, allowing for the removal of the sugar-phosphate residue. DNA polymerase then fills in the gap with the correct nucleotide, followed by DNA ligase sealing the nick to restore the integrity of the DNA strand. This highly coordinated and efficient repair mechanism plays a vital role in preventing genomic instability and maintaining overall cellular function, thereby safeguarding against various diseases, including cancer.
• Homologous Recombination (HR) is a critical process in cellular biology that facilitates the accurate repair of double-strand breaks (DSBs) in DNA. This mechanism relies on the principle of genetic exchange between similar or identical DNA sequences, ensuring that the repaired DNA maintains fidelity to the original genetic code. During HR, the broken ends of the DNA molecule are recognized, and proteins involved in this process, such as Rad51, promote strand invasion into a homologous template, typically the sister chromatid. This invasion allows for the synthesis of new DNA, effectively correcting the damage. HR is especially vital during meiosis, where it enhances genetic diversity by promoting the exchange of genetic material between homologous chromosomes. Additionally, proper functioning of HR is essential for maintaining genomic stability, as failures in this pathway can lead to increased mutagenesis and the development of cancerous cells. Understanding HR's mechanisms and regulations remains a significant focus of genetic research, with implications for gene therapy and cancer treatment strategies.

Hereditary DNA Repair Disorders

• Xeroderma pigmentosum: Characterized by hypersensitivity to UV light, increased skin cancer incidence, and premature aging, due to mutations in genes involved in nucleotide-excision repair (NER).
• Nucleotide-excision repair (NER) is a crucial DNA repair mechanism that removes a wide range of DNA lesions, including those caused by ultraviolet (UV) light and chemical mutagens. This process plays an essential role in maintaining genomic stability by excising damaged DNA segments and accurately synthesizing new, undamaged strands. NER involves a series of coordinated actions, beginning with the recognition of DNA distortion caused by damage. Specialized proteins then detach the segment of DNA surrounding the lesion. After excision, DNA polymerase fills in the gap with the correct nucleotides, and DNA ligase seals the newly synthesized strand into the existing DNA double helix. This complex but highly effective repair system is vital for preventing mutations that can lead to serious consequences, including cancer development.
• Werner's syndrome: A disorder involving premature aging and retarded growth, due to mutations in DNA repair genes.

Mutations

• Mutations are changes in DNA sequence.
• Different types of mutations exist (Point, Chromosome).
• Mutations can be categorized based on the change's nature or consequences (e.g., substitution, frameshift).

Genetic Polymorphism

• A genetic polymorphism represents the presence of two or more relatively common forms of a gene in a population.
• Single nucleotide polymorphisms (SNPs) are common examples, where a single nucleotide differs between forms of a gene.

Test your knowledge on DNA replication processes and repair mechanisms. This quiz covers key concepts such as the models of DNA replication, the significance of the Meselson-Stahl experiment, and the role of mutations in DNA. Dive into the molecular details and enhance your understanding of this fundamental topic.