BIOLOGY

DNA Replication Mechanisms

• Three possible ways to replicate DNA:

  • Semiconservative: Parent DNA splits and serves as a template for a new strand. Each new DNA molecule has one old and one new strand.

  • Conservative: The original parent DNA remains intact while two new daughter strands are created, identical to the parent.

  • Dispersive: Parent DNA is fragmented, and both daughter strands contain segments of the parent molecule as a mosaic.

• Meselson-Stahl Experiment:

  • Demonstrated that DNA replication is semiconservative by using radioactively labeled nitrogen atoms.

  • Heavier, labeled nitrogen provided evidence of the DNA density post-replication in E. coli.

DNA Replication Steps

• Key stages of DNA replication:

  1. Initiation

  2. Elongation

  3. Termination

• Nucleotide Addition: New nucleotides are always added to the 3' end, with DNA synthesis occurring in the 5' to 3' direction. The template strand is read in the 3' to 5' direction.

• Origin of Replication:

  • Prokaryotes have ONE origin of replication, whereas eukaryotes have MULTIPLE origins.

Prokaryotic vs. Eukaryotic DNA

• Prokaryotic DNA:

  • Circular structure

  • DNA helicase separates strands at the origin of replication, forming a replication bubble and fork.

• Eukaryotic DNA:

  • Linear structure

  • Needs multiple origins of replication where the strands separate.

Enzymes Involved in DNA Replication

• Helicase:

  • Creates replication fork by unwinding DNA and separating nitrogenous bases.

• Single-Strand Binding Proteins:

  • Stabilize the single-stranded DNA formed after helicase activity.

• Topoisomerase:

  • Prevents tangling of DNA during replication.

Primer and DNA Polymerase

• Primase:

  • Adds an RNA primer initiated by DNA polymerase.

• DNA Polymerase:

  • Cannot initiate synthesis of new strands without a primer; it binds to the primer-template junction and synthesizes new DNA strands in the 5' to 3' direction.

Elongation Phase

• Top Template Strand:

  • DNA polymerase binds and adds nucleotides.

• Lagging Strand Formation:

  • More complex as DNA polymerase works in the opposite direction of the replication fork.

  • Multiple RNA primers create short DNA sections known as Okazaki fragments, later connected by DNA ligase.

Finalization of DNA Synthesis

• DNA Polymerase I:

  • Replaces RNA primers with DNA.

• DNA Ligase:

  • Forms phosphodiester bonds to connect Okazaki fragments.

• Termination Challenges:

  • Replication ends when the last primer reaches the end of the chromosome. There is no mechanism to replace this primer with DNA, risking loss of essential genetic material.

Telomeres and Aging

• Telomeres:

  • Non-coding sequences at chromosome ends that prevent loss of crucial genes during replication.

  • Shorten with each division, which is significant for cell life and division capacity.

• Telomerase:

  • An enzyme that adds repetitive sequences to telomeres and is active in stem and cancer cells; not present in most somatic cells.

• Aging Relationship:

  • Shorter telomeres are associated with age-related diseases.

DNA Proofreading and Repair Mechanisms

• Proofreading Function of DNA Polymerase:

  • Corrects 99% of errors made during DNA synthesis.

• Mismatch Repair:

  • Fixes errors that escape proofreading by replacing incorrect bases and surrounding areas.

DNA Damage and Repair

• UV Light and Skin Damage:

  • UV exposure can cause DNA damage and mutations; unrepaired DNA may lead to cancer.

• Excision Repair Mechanism:

  • Damaged DNA is excised and replaced by DNA polymerase adding new bases complementary to the template strand.

Central Dogma of Molecular Biology

• Stages of gene expression:

  1. Transcription: Information from DNA is copied into mRNA.

  2. Translation: mRNA guides the synthesis of proteins.

• Transcription Differences in Prokaryotes vs. Eukaryotes:

  • Prokaryotes: Both transcription and translation occur in the cytoplasm.

  • Eukaryotes: Transcription occurs in the nucleus; processing occurs before mRNA is translated into proteins.

RNA Types and Functions

• mRNA:

  • Messenger RNA used in protein synthesis.

• rRNA:

  • Ribosomal RNA, structural component of ribosomes.

• tRNA:

  • Transfer RNA, helps translate mRNA sequence into a protein.

Transcription Process in Eukaryotes

• Promoter Recognition:

  • RNA polymerase binds to the promoter region of the gene, often characterized by A-T-rich sequences for easier binding.

• Transcription Factors:

  • Proteins that assist in the binding of RNA polymerase to DNA at the promoter region.

Gene Regulation in Eukaryotes

• Transcription Factors:

  • Ensure appropriate gene expression by modifying DNA structure and accessibility.

• Epigenetics:

  • Mechanisms such as histone modification that regulate gene accessibility, affecting which genes are expressed without changing the DNA sequence.

Histone Modifications and DNA Access

• Acetylation:

  • Addition of acetyl groups relaxes DNA structure, enhancing transcription accessibility.

• Methylation:

  • Addition of methyl groups can silence gene expression; methylation patterns are stable and can be inherited.

Gene Expression Control Mechanisms

• Alternative Splicing:

  • Produces different proteins from the same mRNA transcript, influencing protein diversity.

• RNA Degradation:

  • mRNA can be degraded by nucleases when no longer needed; protective caps can enhance mRNA stability.

MicroRNA and Translation Regulation

• MicroRNA:

  • Small RNA molecules that can degrade mRNA or inhibit its translation based on sequence complementarity.

• Initiation Factors:

  • Proteins that are essential for the initiation of translation; phosphorylation can inactivate them, halting protein synthesis.

Post-Translational Control

• Ubiquitination:

  • A process where ubiquitin binds to proteins, signaling them for degradation by proteasomes, thus regulating protein levels in the cell.