Lecture 6: Notes_Homologous Recombination
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Homologous Recombination
Homologous recombination is used for experimental techniques like creating transgenic organisms and cell lines to study gene function.
In nature, it repairs large chromosome regions to maintain genome stability and mixes genes during meiosis to generate diversity.
Recommended reading: Molecular Biology of the Cell chapter on DNA repair and meiosis (7th edition, pages 296-306) for detailed understanding.
Definition: Exchange of genetic material between homologous chromosomes to repair DNA and create genetic variation.
Homologous chromosomes are not always identical (e.g., chromosomes from mom and dad) but encode for similar genes, allowing for recombination.
Identical homologous chromosomes exist during mitosis when sister chromatids are attached, ensuring accurate DNA replication.
Gene conversion occurs when a non-identical homologous chromosome repairs a disrupted chromosome, leading to allele conversion.
Biological Uses of Homologous Recombination:
Repair of double-stranded breaks to maintain genome integrity.
Repair of stalled replication forks to prevent DNA damage.
Meiosis to generate genetic diversity during sexual reproduction.
Experimental Applications of Homologous Recombination:
Generating transgenic organisms and cell lines to study gene function and disease.
Transgenic cell lines: e.g., human cell line expressing jellyfish-derived GFP for visualizing cellular processes.
GFP (Green Fluorescent Protein) was first isolated from jellyfish, used as a marker in biological studies.
Generating embryonic stem cells for transgenic mouse technology to create animal models of disease.
Transgenic mice: Gene of interest is introduced into mouse embryos to study its effects on development and physiology.
Transgenic vs. Chimera
Chimera: An organism composed of two distinct genomes, often used in creating transgenic organisms; the initial mouse is a chimera.
Transgenic: An organism that contains genetic material from another species, stably integrated into its genome.
CRISPR-Cas9
Alternative method for introducing genes without relying solely on homologous recombination, offering increased precision.
CRISPR-Cas9 Enzyme: An enzyme with a guide RNA strand complementary to the target region for removal, enabling targeted gene editing.
Function: Generates double-stranded breaks at specific locations in the genome.
Natural Repair Mechanism: Non-homologous end joining, error-prone and can introduce mutations.
Enhanced Precision: Pairing with a homologous region encourages homologous recombination over non-homologous end joining, improving accuracy.
Homologous chromosomes are typically in different nuclear neighborhoods to prevent spontaneous recombination, maintaining genome stability.
Base Pairing Rules
Non-productive Interactions: Initial collisions between complementary strands without stable base pairing.
Productive Zippering: Nucleation occurs, with hydrogen bonds forming, leading to stable double-stranded molecule generation.
DNA Replication and Homologous Recombination
During DNA synthesis, homologous recombination is crucial for repairing replication errors.
Event: Nick occurs on one strand during DNA replication, leading to potential instability.
Issue: Leading strand faces dissociation, disrupting continuous replication.
Solution: Homologous region is utilized to restart replication. The lagging strand is completed and fused to the double-stranded break to avoid nucleotide loss.
Exonuclease Activity: Strands are chewed back, forming a three-prime end, preparing for strand invasion.
Importance of the Three-Prime End: Contains a hydroxyl group, which is nucleophilic, allowing it to invade the strand and initiate repair.
The old strand reconnects with the old strand again, and the repaired region is newly synthesized utilizing a copy of that DNA, ensuring fidelity.
The hydroxyl group is essential for adding new nucleotides, facilitating DNA synthesis.
Process Flow:
Three-prime overhang invades the strand, complementing it, initiating repair.
A bubble forms in the strand due to the three-prime overhang, creating a structure for DNA synthesis.
DNA synthesis continues with the leading strand, filling in the gap.
The newly synthesized strand is kept, and the opened strand is cleaved, completing the repair.
The old strand used for repair is then synthesized as a lagging strand, restoring the original sequence.
Proteins Involved in DNA Repair
Critical role of proteins in holding single-stranded DNA to prevent self-complementing and degradation.
RecA/Rad51: Absolutely required for homologous recombination, essential for strand invasion.
Function: Acts after exonuclease activity to interact with cleaved DNA, promoting strand pairing.
Prevents DNA degradation by protecting single-stranded DNA.
Binds tightly in long cooperative clusters to single-stranded DNA, holding single-stranded DNA and double-stranded duplexes together, stabilizing the structure.
Catalyzes DNA synapse between the double helix and the homologous region of ssDNA, facilitating strand exchange.
Rad51 is the eukaryotic name and RecA the yeast name, Rad52 also can bind a single stranded DNA, all involved in homologous recombination.
RecA and Heteroduplex Formation:
RecA or Rad51 essential for holding this complex in place with three strands in order to finish the homologous recombination, ensuring stability.
Essential for catalysis of DNA synapse between double helix and homologous region of ssDNA, promoting strand exchange.
Formation of a heteroduplex, a double helix formed by pairing of two DNA strands that were originally part of two different DNA molecules, key intermediate in repair.
Branch Migration:
The area where invading strand interacts with the opposing strand, crucial for DNA repair.
Movement: Occurs in either direction post-strand invasion, expanding the repaired region.
Zippering Action: Hydrogen bonding-related movement, facilitating base pairing.
Spontaneous Movement: Can occur without aid, driven by thermal energy.
Helicase-Mediated Branch Migration: Requires energy (ATP) to move towards a specific direction, controlled movement.
Protein-Directed Branch Migration:
Helicase facilitates specific directional movement, controlled by protein interactions.
Involves pairing and displacing bases, essential for proper repair.
Invading strand must invade and complement with donor DNA for proper repair, ensuring accurate sequence restoration.
Homologous Recombination with Sister Chromatid
Occurs with an identical sister chromatid for seamless repair with the utility of an identical sister chromatid, maintaining sequence fidelity.
Double Stranded Break: Both DNA strands are severed, two three-prime overhangs are made, initiating repair.
Nucleophilic Attack: Three-prime overhang invades sister chromatid, initiating strand exchange.
Synthesis: Template is used to synthesize missing DNA, ensuring accurate replication.
Sister chromatid it is going to be used as a template to copy all of the regions that are missing for the above strand, restoring the original sequence.
The sister chromatid from that invasion invades the sister chromatid and goes back, and reinvades the original strand, completing the repair.
Unusual Experiment Demonstrating Homologous Recombination
Based on a bacterial system with a circular chromosome and multiple gene copies, enabling complex recombination events.
GFP Disruption: A cell has GFP with an interrupting element, blocking fluorescence, but with an Enhancer and promoter for expression, allowing for recombination-dependent expression.
Complete GFP Gene: Another part of the chromosome has the intact GFP gene but lacks an enhancer and promoter, requiring recombination for activation.
Homologous recombination must occur for the cell to express GFP, demonstrating the repair mechanism.
Experiment
Investigates the proteins involved in homologous recombination, elucidating their roles.
Process: RNAi is used to diminish the protein function, a loss-of-function technique to study gene requirements.
By the rules of science to ask about the requirements or function of a gene product lose it experiment is performed, assessing its necessity.
The experiment asks about the, requirement of RAD 51 and RAD 52 essential proteins in homologous recombination.
Terminology
Genetic Knockout (Loss of Function): Eliminating gene expression to study its role.
siRNA Treated Cell (Loss of Function): Reducing gene expression using small interfering RNA.
Cell Treated with Inhibitor (Loss of Function): Blocking protein function using chemical inhibitors.
siRNA Efficacy
Effective siRNAs: Target mRNA for degradation or block translation, reducing protein levels.
Maximum Effectiveness: siRNAs are typically 70% effective since siRNAs are not perfect, accounting for incomplete knockdown.
Cell Line Specificity
Cell Lines and siRNA: Different cell lines (e.g., Hep293, U2OS) react differently to siRNAs, affecting experimental outcomes.
Cell Line as N of One: Each cell line is a single genome, and differences indicate real phenomena, underscoring biological variability.
Different cell lines are needed to create more ends, increasing statistical power.
Observations
Knockdown Effects: Loss of function experiments with siRNA against Rad51, Rad52, assessing their impact.
Rad51 and BRCA2: Required for homologous recombination, playing essential roles in DNA repair.
BH1: May inhibit homologous recombination; its absence enhances recombination, suggesting regulatory roles.
DRGFP Explanation
DRGFP indicates that in the genome of these cells, there is a defective GFP that can be only expressed through recombination, serving as a reporter.
Homologous Recombination in Meiosis
Meiosis: Homologous Recombination occurs during meiosis to generate gametes for sexual reproduction, creating genetic diversity.
Genetic Testing: Genetic testing is performed to assess alleles for defects and potential for disease, informing reproductive decisions.
The egg and the sperm are both cells, and they're both haploid for a mammalian system, containing half the number of chromosomes.
Egg are huge because they have all the components necessary for cells, supporting early development.
Functions of Gametes
Sperm: Designed to deliver a haploid genome across long distances in an aquatic environment, optimized for motility.
Egg: Has essential components to support life and initiate gene expression (nucleotides, ATP, organelles, etc.), ensuring early development.
Homologous Recombination During Meiosis Essential to have homologous recombination in, the generation of gametes for sexual reproduction, promoting genetic diversity.
Genome Mixing to Protect Against Disease from Mutation and Repair Potential, enhancing adaptability.
There are genes in both parents that can be effective and play a role in repairing diseases, contributing to offspring health.
Homologous Chromosomes: Homologs recognize, pair, and segregate into separate cells, exchanging a portion of DNA. This recombination creates genomic diversity, increasing adaptability.
Mother has both paternal and maternal. Recombination creates unique code that we acquire for our parents, leading to diverse traits.
50/50 Genetic Contributions (Excluding Mitochondrial DNA): More genetic material comes from the maternal contribution due to maternal inheritance of mitochondria, impacting cellular functions.
Not fully understood how, but homologous recombination does occur in all types of conditions, highlighting its robustness.
Meiosis
Homologous chromosomes are paired only due to meiosis for recombination to take place, a unique feature of sexual reproduction.
The sister chromatids recombine with the other parents homologous chromosomes yielding four different combinations of these homologous chromosomes, increasing genetic diversity.
Stages of Prophase I in Meiosis
Leptonema: Condensation of homologous chromosomes, initiating meiosis.
Zygonema: Chromosomes cross over but have not yet recombined (poised for recombination), forming the synaptonemal complex.
Pachyneema: Recombination occurs, breaking is done in single stranded breaks or double stranded breaks, and invasion occurs, repairing and creating diversity.
Diplonema: Regions where crossing over occurs (chiasmata) are visible, holding chromosomes together.
Understanding Chromosome Structure
Sister Chromatids (2 copies each from the maternal and paternal homologous chromosomes), ensuring genetic redundancy.
Synaptonemal Complex
Proteins known as synaptonemal complex proteins holds these chromosomes, facilitating recombination.
Composed of Synaptonemal complex one: transverse filament, SEP2, which consists of actually lateral elements, SEP three and another lateral element, forming the structural support.
Gene Function Experiments
First Type of Approach: a loss of function to understand the role of a protein, the importance of a protein, revealing its necessity.
If One Removes the Protein, and There Is No Phenotype? : If there Is, is to experiment with a gain of function movement experiment. You can overexpress the protein to see if we can derive a function or sufficient ability, complementing the loss-of-function analysis.
SEP 3 Functions
Not interacting with the chromosomes at leptotene as robustly, but by the electron telling you notice that it becomes vigorous because the clusters of proteins start condensing more, indicating its regulation.
Proteins become more robust, stabilizing the synaptonemal complex.
At the leptotene stage there is a possibility expression is increased, adjusting its function.
In zygotene you can see these strips. A GFP has clustered onto these homologous chromosomes, but they have not yet combined to each other at the packeting so that we see less the chromosomes since most cells are together in colocolization, visualizing the dynamics.
In mutants the the sister chromatids have not adhered, disrupting chromosome structure.
sEP one is lost which transverse protein that leads to the homologous not being closely together, impairing recombination.
SPO-11
In SPO 11 minutes you don't get the recombination, and they stay arrested at the zygotene stage, blocking meiosis.
There is no separation as to where proteins begin to interact, disrupting chromosome dynamics.
Resolution
Resolution of meiotic exchange also known as holiday junction, completing recombination.
Heterologous Recombination : Where one strand copies, and then it returns so only a portion of the one homologous chromosome gets coppied, resulting in gene conversion.
Heterologous Recombination 2 Crosses:. where it copies, and then each chromosome copies one another to allow something known as gene conversion, expanding sequence diversity.
Ninety Percent of Spo11 Events Are Resolved as Not Crossovers- where one homologous copied strand event happens, regulating recombination outcomes.
In Crossovers, not only is it just one side copying another, but both homologous chromosomes are recombining, generating new combinations.
Not clear whether crossover is selected or not. Two crossovers occur during meiosis, ensuring genetic diversity.
In Gene Conversion the homologous chromosomes mismatch, but one gets selected for such