Genetic Recombination PDF

**Genetic Recombination** **What is Recombination?** - Breaking and rejoining DNA in new combinations -   **Two Types of Recombination** - **Homologous recombination** -  - Involves exchange of DNA segments between two DNA molecules -...

**Genetic Recombination** **What is Recombination?** - Breaking and rejoining DNA in new combinations -   **Two Types of Recombination** - **Homologous recombination** -  - Involves exchange of DNA segments between two DNA molecules - Highly similar or identical sequences - Requires extensity sequence similarity (homology) between segments - Uses general recombination proteins - Bacteria - RecA - Eukaryotes - o51 - Also uses helicases/nucleases like RecBCD to process DNA - Biological functions - Repair of double-stranded DNA breaks - Ensures genetic diversity during meiosis by crossing over - Maintains genome integrity - Outcome - Results in an exchange between homologous regions - Often leads to crossover products - **Site-specific recombination** - specific sequence - Involves exchange of DNA at specific recognition sequences - Typically short and unique sequences - Requires a specific short DNA sequence - Recognised by recombinase enzymes - Does not depend on extensive homology - Relies on recombinase enzymes (Cre (Cre-Lox system)) or integrases (λ integrase) - Biological Functions - Integration of viral genomes into host DNA - E.g. bacteriophage λ integration into E. coli - Gene regulation - E.g. inversion of DNA segments - Use in genetic engineering - E.g. Cre-Lox for precise DNA modifications - Outcome - Typically leads to insertion, deletion or inversion of DNA segments - Depends on the orientation and position of recognition sites   **Recombination in Meiosis I** -    **Sister Chromatid Exchange (SCE)** - Involves exchange of genetic material between two identical sister chromatids - Occurs in mitosis as a repair mechanism - Resolves DNA damage or replication errors - Technically a recombination event - Does not lead to genetic diversity as the chromatids are identical - Occurs between template DNA strand and newly synthesised strand after DNA replication - **Visualising (Harlequin Chromosomes)** - Cells undergo two rounds of DNA replication - In first round, bromodeoxyuridine (BrdU) substitutes partially for thymidine in newly synthesised strands - In second replication, chromosomes contain one BrdU-labelled strand and one unlabelled strand - When stained with a specific dye, they have alternating dark and light patterns due to sister chromatid exchange -   **Summary so far** - Genetic recombination is the breaking and rejoining of DNA in various patterns - The key differences between recombination in meiosis and mitosis are: - In meiosis, recombination occurs between non-identical chromatids, while in mitosis, recombination occurs between identical sister chromatids - In meiosis, recombination is vital for increasing genetic diversity, potentially allowing for greater heterozygosity and potential for adapting to changing environments or stresses by making sure that each gamete is unique. In mitosis, sister chromatid exchange is essential for ensuring that daughter cells are genetically identical by fixing DNA damage or replication mistakes, and creating overall greater genomic stability **Holliday Junctions**  **What is the Holliday Junction?** - - Four-stranded DNA structure that forms when: 1. Two double-stranded DNA molecules align - Homologous or nearly identical 2. One strand from each DNA molecule is nicked, then invades the other DNA molecule - Pairs with its complementary strand 3. Creates a crossover point where two single DNA strands exchange partners - Forms a cross-shaped structure   **Formation Process** 1. Single-strand breaks - Two homologous DNA molecules experience single-strand nicks at similar positions 2. Strand invasion - Each nicked strand invades the other DNA molecule pairing with complementary strand - Forms a heteroduplex region 3. Crossing over - Invading strands are joined to opposite duplex - Forms Holliday junction 4. Branch migration - The junction can move along DNA via a process called branch migration - Extends the region of exchanged strands 5. Resolution - Holliday junction is resolved (cleaved) by specific enzymes - Bacteria - RuvC - Eukaryotes - GEN1 - Can result in two outcomes - Crossover products - chromosomes exchange large DNA segments - Non-crossover products - DNA strands are restored without significant exchange of genetic material   **Key Features** - Four strands - Two DNA duplexes are connected at the junction point - Symmetry - Junction is initially 4-fold symmetrical - Can adopt a more stacked-X formation - Two strands are antiparallel and stacked - Dynamic nature - Branch migration allows the junction to move along the DNA - Changes the length of the heteroduplex region   **Folding** -    **Stacked-X conformation** - - Holliday Junction adopts this conformation in physiological conditions - Such as in presence of divalent cations like magnesium (Mg^2+^) - Features - Stacked base pairs - The arms of the DNA are folded and stacked - Forms cross-like shape when viewed from the side - Increases stability of junction - Two-fold symmetry - Stacked-X breaks the original four-fold symmetry of Holliday junction - DNA arms form two sets of continuous helices - Strands exchange partners in an antiparallel orientation - Two types of strands - Continuous - Exchanging   **Alternative Conformers** -  - **Yes**   **Branch Migration** - Process of a crossover point (branch) of a DNA Holliday junction moves along the DNA duplex - **Steps** 1. Formation of Holliday junction 2. Movement of the branch - The crossover shifts along the DNA - Happens as base pairs are sequentially broken and reformed further down the duplex in a coordinated manner 3. Extension of heteroduplex DNA - Region where DNA strands from different molecules are paired (heteroduplex) grows or shrinks depending on the direction of branch migration - **Spontaneous branch migration** 1. Occurs naturally due to thermal fluctuations 2. Typically slow and limited as DNA base-pairing stabilises the junction - **Protein-assisted branch migration** 1. RuvA and RuvB - bacteria - Form a molecular motor that unwinds and moves junction along DNA 2. RAD51 and associated proteins - eukaryotes - Facilitate strand pairing and heteroduplex extension - **Roles** 1. Meiotic recombination - Extends heteroduplex region - Essential for genetic diversity and adaptation 2. DNA repair - Important in homologous recombination repair of double-strand breaks - Helps in the precise alignment and repair of damaged DNA 3. Genome stability - Efficient branch migration ensures accurate repair and recombination - Maintains genetic integrity   - One strand - - Two strands -      **Double-strand break repair (DSBR) model** - 1. Double-strand break (DSB) - Double-strand break occurs in one of the DNA molecules - Can be due to damage - Can be programmed breaks to initiate recombination process - Initiates homologous recombination 2. Processing the break - Exonuclease activity degrades the DNA at the break - Removes nucleotides from 5\' ends on both strands - Creates single-stranded 3\' overhangs (ss 3\' ends) 3. Strand invasion and D-loop formation - One of the ss 3\' overhangs (green) invades the homologous double-stranded DNA molecule (red) - Pairs with its complementary strand - This forms a D-loop (displacement loop)where the displaced strand of the red DNA is pushed out - Invading strand uses this complementary strand of homologous DNA as a template for DNA synthesis 4. Repair synthesis - DNA polymerase uses this template to extend the invading strand - Displaced strand in the D-loop can also serve is a template for synthesis of the second strand (not shown) 5. Formation of Holliday junctions - Apter repair synthesis, displaced strand pairs with the other 3\' overhang on broken DNA - Leads to formation of two Holliday junctions 6. Resolution - Two Holliday junctions are resolved by specific enzymes (resolvases) - Final products can be crossover products or non-crossover products - Depends on how the junctions are cleaved - This can happen in non-dividing cells - Homologous recombination can use a homologous sequence to repair DSBs - Sister chromatid, homologous chromosome etc. **Homologous Recombination in E. coli**    **Pre-junction formation**   **Creation of recombinogenic end** - RecBCD - Nuclease and helicase complex - Moves along DNA - RecB - 3\'-5\' helicase and nuclease - Beta chain - RecD - 5\'-3\' helicase - Gamma chain - RecC - recognises Chi sequence - Alpha chain - Crossover Hotspot Instigator sites - Regulates nuclease activity - Step-by-step - Binding to double-strand breaks - RecBCD complex binds to broken end of a double-stranded DNA molecule - Unwinding DNA - Helicase activities of RecB and RecD unwind DNA at a high speed - Creates two single-stranded tails - Degradation of DNA - Initially, the nuclease activity of RecB degrades both strands of DNA as it unwinds - Ensures that damaged or unwanted DNA near the break is removed - Removes any damaged, mismatched or unwanted sequences near site of DSB - Exposes - Recognition of Chi sites - As the complex moves along the DNA, RecC scans for Chi sequences - 5\'GCTGGTGG-3\' - These are hotspots for recombination - When a chi site is encountered on the 3\' strand - RecC binds to Chi sequence, pausing the complex - Chi site alters RecBCD\'s behaviour, reducing degradation of 3\'strand and increasing degradation of 5\' strand - Creation of 3\' overhang - After Chi recognition, RecBCD modifies its nuclease activity - Creates a 3\' single-stranded overhang - Overhang is crucial for strand invasion during homologous recombination - Loading of RecA - RecBCD facilitates the loading of RecA protein onto 3\' overhang - RecA promotes strand invasion, forming D-loop structure and initiating homologous pairing   **Strand Invasion**  - RecA - Binds to ssDNA to generate a filament - Active species in strand exchange   - Filament - Contains 1 monomer RecA per 4-9 nt DNA in stable complex - Polar - binds in 5\'-3\' direction - i.e. migrates to 3\' end of molecule - SSB facilitates process - Removing secondary structure - ATP not required to form - Increase in ATP increases rate of dissociation - DNA extended in filament by factor 1.5 - 2 - In vitro RecA promotes homologous pairing between various substrates - Nucleoprotein Filament - Helical structure with one RecA monomer per 4-9 nucleotides - Stabilises the ssDNA, keeping an extended conformation suitable for homology searching and strand exchange - Stretches DNA by 1.5-2x normal length - ATP hydrolysis regulates filament disassembly - Ensures controlled activity - Role of Filament - Homology search - RecA filament scans the dsDNA for regions of sequence complementarity to the ssDNA - Forms transient triplex structures (ssDNA + dsDNA) to test for homology - Strand exchange - Once homology is identified, the filament promotes the exchange of invading dsDNA with one strand of dsDNA - Forms initial heteroduplex - Facilitation of repair - Filament ensures precise alignment and repair by using undamaged homologous DNA as a template - Role of ATP - ATP binding - Required for RecA to polymerise on ssDNA and form the filament - Stabilises the filament during homology search and strand exchange - ATP hydrolysis - Drives disassembly of filament after strand exchange is complete - Ensures process is tightly regulated and reversible if needed - Summary of functions **Function**   **Mechanism** ------------------------ --- ------------------------------------------------------ **Homology Search**   Aligns ssDNA with homologous dsDNA. **Strand Invasion**   Promotes D-loop formation and heteroduplex creation. **DNA Repair**   Assists in repairing DSBs and ssDNA gaps. **Filament Formation**   Extends and stabilises ssDNA for recombination.   **Branch Migration** - RuvAB - RuvA - DNA binding protein - Binds selectively to DNA junctions as a tetramer - RuvB - ATPase - Binds to DNA as a hexamer - Exhibits DNA helicase activity - Actions of RuvA and RuvB on a junction - RuvA recognises Holliday junction - RuvA specifically binds to 4-stranded DNA (Holliday junction - Forms a tetramer that clamps onto the junction - Holds junction in open and planar conformation but stably\\ - Recruitment of RuvB - RuvA acts as a docking platform to recruit RuvB - Assembly of RuvB rings - RuvB hexameric rings assemble on the DNA duplexes flanking the Holliday junction - Rings encircle DNA strands near the junction - Positioning of DNA - RuvA ensures proper alignment of the DNA strands within the junction - Positions strands for efficient branch migration by RuvB - ATP binding and hydrolysis - RuvB hydrolyses ATP, generating the energy needed to pull DNA through its hexameric ring structure - Drives movement of crossover point along DNA duplex - Two RuvB rings (one on each end of junction) - One pulls DNA while one stabilises and acts as secondary motor - Ensures branch migration occurs in one direction - Branch migration - Coordinated action of the two RuvB rings pulls DNA in opposite directions - Crossover point migrates along the DNA - Extends or reduces heteroduplex region - Continuous guidance by RuvA - RuvA remains bound at the Holliday junction to keep DNA aligned and maintain proper geometry for efficient migration - Preparation for resolution - Once Holliday junction has migrated to desired position, RuvA and RuvB position it for cleavage by RuvC - RecG - Alternative to RuvAB - Functions of RecG - Specifically recognises Holliday junctions - Binds to Holliday junctions using helicase domains - Bind to all 4 strands and - Is uniquely suited to recognise and stabilise the crossover point - Positions DNA for migration - Unwinds and Migrates DNA - Uses helicase activity to unwind DNA - Drives branch migration of Holliday junction - Driven by ATP hydrolysis - Acts as a single-protein helicase - Does not require additional motor proteins like RuvB - Directionality - Typically migrates junction in a direction that restores the original DNA duplexes - Prepares DNA for resolution - RecG instead of RuvAB? - RecG often acts as a backup mechanism when RuvAB pathway is unavailable or insufficient - RecG has different substrate preferences and may function in situations where RuvAB is less efficient   **Resolution**  - Junction resolving enzymes - Structure-selective nucleases - Targeted to DNA junctions - Resolution reaction - RuvC - Resolvase enzyme - Functions as a dimer - As Each contain an active site, they can act on both DNA strands at the same time - Actions of RuvC - Recognition of Holliday junction - Recognises unique 4-stranded structure of the Holliday junction - Binds the junction, often in cooperation with RuvAB complex - Junction is positioned for cleavage - Stabilises Holliday junction and prepares it for enzymatic resolution - Cleavage of Holliday Junction - RuvC is a structure-specific nuclease that introduces two symmetrical cuts in the DNA strands - It cleaves opposing strands at the crossover point of the Holliday junction - Often cleaves at a specific consensus sequence within the junction - Can also resolve junctions at other sites - Does not require ATP hydrolysis - Relies solely on nuclease activity - Formation of two separate DNA molecules - After cleavage, Holliday junction is resolved into two distinct DNA duplexes - Type of product formed depends on orientation of cuts - Crossover products - Large segments of DNA are exchanged between two molecules - Increase genetic diversity - Non-crossover products - Two molecules are restored to their original configurations without large-scale exchange **Homologous Recombination in Yeast** **5 Key Steps**   **Double Strand Break an Overhang Formation** - MRX complex moves to site of break - Proteins - RAD50 - Coiled-coil structure - Tethers broken DNA ends - Ensures DNA ends remain aligned and in proximity - ATPase domain - XRS2 (NBS1 - Helps localise MRX complex to sites of DNA ds breaks (DSBs) - Interact with DNA damage response proteins - Helps initiate DNA resection - Degrading 5\' ends of DNA to produce 3\' overhangs - Plays a role in activating DNA damage checkpoints - Ensures repair occurs before cell cycle progression - MRE11 - Nuclease activity - Creates nicks near DSB to facilitate processing - Removes nucleotides from DNA ends - Initial resection of 5\' ends - Works with Sae2 (CTIP in humans) - Activates Tel1 - Triggers DNA damage response - MRX complex initiates recombination - With help from Sae2 - 5\' DNA is degraded, leaving a 3\' overhang - Overhang is bound by replication protein A (RPA), stabilising the ssDNA - Displaced by RAD51   **Filament creation** - Single-stranded overhang is bound by RAD51 - With help of RAD52 (BRAC2 in humans (tumour suppressor)) - Creates nucleoprotein filament - Essential for homology search and strand invasion   **DNA replication** - DNA polymerase delta uses the duplex as a template to replicate the single stranded DNA - Ligase fills the gaps   **Formation of Holliday junction** - Second-end capture   **Resolution of Holliday junctions** - Cleavage of Holliday junction by structure-specific endonucleases (resolvases) - Resolvases - GEN1 - Binds to Holliday junction in open-planar conformation - Introduces two symmetrical nicks in opposing strands of DNA