Homologous Recombination

• Lecture 9 is about homologous recombination
• The lecture date is March 17, 2025

Homologous Recombination Steps

• DNA duplexes are cleaved by Spo11, resulting in a double-strand break with Spo11 covalently attached.
• Spo11 is released via endonucleolytic cleavage by MRX (Mre11, Rad50, Xrs2) and Sae2, liberating Spo11 attached to a short oligonucleotide.
• 5' strands are resected by 5'-3' exonucleases (Exo1 in yeast), creating long single-stranded tails coated with ssDNA-binding protein RPA (Replication Protein A).
• RPA is replaced by recombinases Rad51 and Dmc1, forming a nucleoprotein filament for sequence similarity search on the homologous chromosome, creating D-loop structures.
• Following DNA synthesis, recombination structures undergo one of two main outcomes.
• An invading strand is ejected by helicases, allowing DNA ends to re-anneal in a process called synthesis-dependent strand annealing,producing non-crossovers, with local transfer of genetic information.
• Alternatively, recombination structures are stabilized by "ZMM" proteins and channeled through the double Holliday Junction (dHJ) resolution pathway, which mostly produces crossovers.
• Both break ends engage the donor to form a double Holiday Junction intermediate, which is resolved through a crossover-specific pathway involving MutLγ and Exo1.

Axial Elements in DSB Formation

• The chromosome axis in yeast contains a cohesin complex with the meiosis-specific kleisin subunit Rec8, Hop1, and the core axial protein Red1.
• Deletion of Red1 causes a 2.5 to 5-fold reduction in DSB formation, while Hop1 deletion decreases DSB levels by at least 10-fold emphasizing the importance of axis proteins for DSB formation.

Recombination in Bacteria

• Bacterial recombination involves restricted regions of DNA molecules rather than intact chromosomes.
• Molecular events involve a single strand from a broken molecule interacting with a partner duplex, extending the pairing region, and an endonuclease resolving the partner duplex.
• Bacteria typically exchange limited amounts of duplex DNA, but prokaryotes can initiate recombination through various routes.
• DNA may be available with free single-strand 3' ends or provided in single-stranded form, like in conjugation.
• Single-strand gaps can result from irradiation damage, or single-stranded tails from phage genomes undergoing replication by a rolling circle.
• Key features include exchange of sequences, recognition reactions involving restricted DNA regions, and a similar order of events as in other organisms

Chi Sequences

• Chi sequences are hotspots stimulating recombination in bacteria.
• Mutants discovered in phage lambda called chi have single base-pair changes that stimulate recombination.
• A chi sequence stimulates recombination within approximately 10 kb.
• A chi site is activated by a DSB several kb away, requiring the recombination apparatus to associate with DNA at a broken end and move along the duplex in one direction.
• Chi sites are targets for the recBCD enzyme, whose activities include nuclease activity to degrade DNA, helicase activity to unwind duplex DNA in the presence of SSB protein, and ATPase activity.
• The enzyme's role in recombination may be to provide a single-stranded region with a free 3' end.

The Bacterial RecBCD System and Chi Sequences

• The RecBCD complex possesses both nuclease and helicase activities.
• RecBCD binds to DNA downstream of a chi sequence, unwinds the duplex, and degrades one strand from 3'-5' as it moves to the chi site.
• The chi site triggers the loss of RecD subunit and affects nuclease activity.
• RecBCD is involved in replication fork recombination events.

Strand-Transfer Proteins

• RecA forms filaments with single-stranded or duplex DNA and catalyzes the ability of a single-stranded DNA with a free 3' end to displace its counterpart in a DNA duplex.
• Presynaptic filaments consist of single-stranded DNA bound in a helical nucleoprotein filament with a strand transfer protein such as Rad51 or RecA.
• The DNA handling activity of RecA displaces its homolog in a duplex in a reaction called single-strand assimilation (or invasion).
• The displacement reaction conditions include one DNA molecule having a single-stranded region, one having a free 3' end, and the single-stranded region and 3' end needing to be complementary between the molecules.

Holliday Junctions

• The bacterial Ruv complex acts on recombinant junctions, with RuvA recognizing the junction's structure.
• RuvB, a helicase, catalyzes branch migration.
• RuvC cleaves junctions to generate recombination intermediates.
• Resolution in eukaryotes is understood less, but several meiotic and mitotic proteins are involved.
• Patch recombinants are DNA resulting from a Holliday junction being resolved by cutting exchanged strands.
• The duplex remains largely unchanged, except for a DNA sequence on one strand originating from the homologous chromosome.

Eukaryotic Genes in Homologous Recombination

• The MRX complex, Exo1, and Sgs1/Dna2 in yeast, along with the MRN complex and BLM in mammalian cells, resect double-strand breaks.
• The Rad51 recombinase binds to single-stranded DNA with the help of mediator proteins, overcoming the inhibitory effects of RPA.

Presynapsis

• In mitotic cells, DSBs are produced due to exogenous sources or endogenous sources, during replication the nicks are processed into DSBs, which generates single stranded tails with 3’OH ends.
• In meiosis, DSBs are induced by Spo11 dependent cleavage.
• Binding of broken ends by the MRN or MRX happens along with the endonuclease Sae2 (CtIP in mammalian cells).
• Mre11 occurs with (Rad50, Xrs2 in yeast) or with (Rad50 and Nbs1 in humans).
• Rad50 is implicated in the process of holding broken ends together.
• Mre11, Rad50 and Xrs2 mutations result in cells becoming sensitive to ionizing radiation, and diploids have poor miotic outcome.

Synapsis

• A search for homology with other DNA molecules begins after the Rad51 filament has formed on ssDNA, strand invasion results in the development of D-loop.
• Strand invasion needs the action of Rad54 and Rdh54/Tid1 ( in yeast) and RAD54B in mammalian cells.
• Rad54 and Rdh54 are SWI/SNF chromatin remodeling superfamily members with dsDNA dependent ATPase activity.

DNA Heteroduplex Extension and Branch Migration

• Homologous DSBR and SDSA pathways have D-loop extension
• D loop formation leads to the arrangement of Rad51 filament on dsRNA
• Rad54 can remove Rad51 from dsDNA for DNA Polymerase by extending it from the 3' end

Eukaryotic Genes in Homologous Recombination

• Strand invasion depends on Rad54 and Rdh54 in yeast and Rad54 and Rad54B in mammalian cells.
• Yeast Sgs1 and Mus81/Mms4 and human BLM and MUS81/EME1 resolve holliday junctions.