Genet 270 lec 10- recombination pt 1
What is recombination in simple terms?
Recombination is the process where two DNA molecules are broken at the same position and then rejoined in new combinations, resulting in an exchange of genetic material between the molecules.
🧠 Tip: “Break and rejoin — mix the code, not just copy it.”
What is the main purpose of homologous recombination in bacteria?
To promote evolution and genetic diversity by creating new gene combinations and enabling DNA repair and genome rearrangements.
🧠 Tip: “Recombination = innovation + repair.”
How does recombination contribute to bacterial evolution?
Changes gene order through deletions or inversions.
Moves genes to different replicons (chromosome ↔ plasmid ↔ phage DNA).
🧠 Tip: “Recombination reshuffles — inside and between genomes.”
Give examples of recombination events that move genes between replicons.
Conjugation: recombination between an Hfr donor and F⁻ recipient.
Transduction: integration after generalized or specialized phage transfer.
Plasmid integration: plasmid DNA recombines into the chromosome (Hfr formation).
Phage integration: phage DNA integrates into the host chromosome (lysogeny).
🧠 Tip: “Conjugation, transduction, integration — three highways of recombination.”
Besides evolution, what is another major purpose of recombination in bacteria?
Facilitation of DNA replication, especially for DNA repair and restart of collapsed replication forks.
🧠 Tip: “Recombination rescues replication.”
Why is homologous recombination important for DNA replication survival in bacteria?
Bacteria have only one origin of replication (ori) per chromosome.
A nick or break in the template strand can halt replication.
Homologous recombination provides a mechanism to repair or restart these collapsed forks — a key evolutionary advantage.
🧠 Tip: “One ori, one shot — recombination saves the fork.”
What are the two general types of recombination in bacteria, and how do they differ?
Site-specific recombination
Rare
Occurs between short, defined DNA sequences
Requires specialized recombination proteins (e.g., phage integrase, transposase)
Homologous recombination
Common
Occurs between long, similar DNA regions (~20–500 bp)
Uses host recombination machinery (e.g., RecA, RecBCD, RecF)
🧠 Tip: “Site-specific = precise; Homologous = flexible.”
What is a shared outcome of both site-specific and homologous recombination?
Both involve DNA breakage and rejoining, leading to crossovers or integration events in the genome.
🧠 Tip: “Different routes, same result — DNA exchange.”
Who first demonstrated genetic recombination in phages, and when?
Hershey and Rotman (1940) coinfected E. coli with two T2 phages — r⁻h⁺ and r⁺h⁻ — each at a multiplicity of infection (MOI) = 5.
They observed 2% recombinant progeny (r⁻h⁻ and r⁺h⁺), proving phage recombination occurs.
🧠 Tip: “Hershey & Rotman mixed phages, saw recombination miracles.”
What did the Hershey & Rotman experiment demonstrate about phages?
Recombinant genotypes (r⁻h⁻, r⁺h⁺) arose from genetic recombination, not mutation.
Phages could therefore be used as powerful models for genetic analysis.
🧠 Tip: “Phages recombine like chromosomes — perfect genetic tools.”
What were the two early models proposed to explain recombination?
Copy choice model: recombinants form during DNA replication, where the polymerase switches templates between parental strands.
Breakage and reunion model: recombinants form by physical breakage and rejoining of parental DNA molecules.
🧠 Tip: “Copy = template switch; Break = DNA stitch.”
Which of the two historical models better explains recombination in modern terms?
The breakage and reunion model, since recombination is now known to occur through DNA strand breakage, exchange, and rejoining — mechanisms later described by the Holliday model.
🧠 Tip: “Recombination isn’t copy-paste — it’s cut and rejoin.”
What did Meselson and Weigle (1962) aim to test, and in what system?
They tested the breakage and reunion model of recombination using bacteriophage λ.
🧠 Tip: “M&W proved it’s break-and-rejoin, not copy-and-slide.”
How did Meselson & Weigle distinguish parental DNA in their recombination experiment?
Phage DNA grown in N¹⁵ medium → heavy DNA.
Phage DNA grown in N¹⁴ medium → light DNA.
They then coinfected E. coli with heavy (A⁺R⁻) and light (A⁻R⁺) phages to track mixing in progeny using density gradient centrifugation.
What were the results of the Meselson & Weigle experiment?
The recombinant progeny had intermediate densities (ranging from heavy to light), showing that each recombinant phage contained DNA from both parents.
🧠 Tip: “Mixed DNA density = physical DNA exchange.”
How did the Meselson & Weigle results disprove the copy-choice model?
If recombination occurred by copy-choice (template switching), all recombinant DNA would have been uniformly light (since replication occurred in N¹⁴ medium).
The observed range of densities proved that parental DNA physically broke and rejoined, supporting the breakage–reunion model.
🧠 Tip: “Different densities = real DNA breakage.”
What are the main requirements for homologous recombination?
High sequence similarity (≈95%) between recombining DNA regions.
Complementary base pairing between strands of the two DNA molecules.
Involvement of many gene products (e.g., RecA, RecBCD, RecF, Ruv, RecG).
🧠 Tip: “Homology, pairing, proteins — the recombination trio.”
What is the role of sequence similarity in homologous recombination?
Ensures that recombination occurs only between nearly identical DNA regions, preventing loss or mismatch of genetic information.
Typical requirement: ≥95% sequence identity across the crossover region.
🧠 Tip: “High homology = safe exchange.”
What is a synapse in homologous recombination, and how is it formed?
A synapse is the point where two DNA molecules are paired via complementary base pairing between their strands.
Formation requires a mechanism that allows strand pairing between double helices without fully separating them.
RecA protein later facilitates this process.
🧠 Tip: “Synapse = site where DNAs shake hands.”
What types of enzymes are required for recombination, and why?
Recombination requires enzymes that break and rejoin DNA strands, including:
Nucleases → create nicks or breaks in DNA strands.
Ligases → seal new joins between different DNA molecules.
These allow physical strand exchange, similar to replicative transposition.
🧠 Tip: “Cut with nucleases, glue with ligase.”
What is a heteroduplex in recombination?
A heteroduplex is a region within the synapse where base pairing occurs between complementary strands from two different DNA molecules.
All four DNA strands are involved.
Forms after strand invasion and alignment of homologous sequences.
🧠 Tip: “Hetero = mixed; duplex = paired.”
What is the purpose of heteroduplex formation in recombination?
It stabilizes the synapse by allowing hybrid pairing between homologous DNA strands, enabling exchange of genetic information and eventual formation of crossovers.
What are the core shared features of all molecular models of recombination?
All models include:
DNA breakage and rejoining steps,
Heteroduplex formation,
Enzymatic processing by nucleases and ligases, and
Resolution of crossover structures.
Different pathways (e.g., RecBCD, RecF) follow these shared steps.
What is the Holliday model of recombination, and who proposed it?
Proposed by Robin Holliday (1964), the model describes homologous recombination via reciprocal strand invasion:
Two single-strand breaks occur at identical positions in both DNA molecules.
Free ends cross over and base pair with complementary strands of the other DNA.
Ligation forms a cross-shaped (cruciform) structure called the Holliday junction.
🧠 Tip: “Break, cross, join — Holliday’s recombination coin.”
What happens to a Holliday junction after formation?
It can isomerize between two structural conformations.
When cut and religated (resolved), the outcome depends on the orientation of the resolution cut:
One orientation → recombinant (crossover) molecules.
The other → nonrecombinant (patch) molecules.
🧠 Tip: “How you cut decides who swaps.”
What is branch migration in recombination?
Branch migration is the movement of the Holliday junction (the crossover point) along the DNA duplex, extending or shifting the region of heteroduplex DNA.
🧠 Tip: “The crossover travels — junction on the move.”
What enables branch migration to occur?
Requires specialized proteins that break and reform hydrogen bonds rapidly between the base pairs.
This allows the junction to move up or down the DNA without dissociating the duplex.
Expands the heteroduplex region.
🧠 Tip: “Proteins push the branch; DNA keeps swapping partners.”
Is there a mechanism that ensures the same cutting site in both DNA molecules during recombination?
No — there is no precise mechanism ensuring identical strand cutting positions in both DNA molecules.
This lack of precision can lead to mismatched bases or gene conversion within heteroduplex regions.
What is the Meselson–Radding model of recombination?
Proposed by Meselson and Radding, this model describes recombination starting with a single-strand nick in one DNA molecule (not both).
The exposed single strand then invades another double-stranded DNA at a homologous site, initiating strand exchange.
🧠 Tip: “One strand breaks, one strand invades.”
Outline the steps of the Meselson–Radding model.
One DNA molecule experiences a random single-strand cut.
The free strand invades another double-stranded DNA, displacing one strand.
DNA polymerase fills in the gap left by the invading strand.
The displaced strand is degraded.
The new end is ligated, forming a Holliday junction.
The junction undergoes isomerization and resolution.
How does heteroduplex formation differ in the Meselson–Radding model?
Initially, the heteroduplex forms in only one DNA molecule.
Branch migration later extends it into the second molecule, producing the symmetrical structure seen in the Holliday model.
🧠 Tip: “Starts one-sided, migrates two-sided.”
What is the Double-Strand Break Repair (DSBR) model of recombination?
Recombination initiated by a double-strand break (DSB) in one DNA molecule.
Both strands are broken, and recombination repairs the break by using a homologous DNA molecule as a template.
🧠 Tip: “Two strands broken, one partner borrowed.”
Outline the steps of the Double-Strand Break Repair (DSBR) model.
Both DNA strands broken.
5′ ends degraded by exonuclease → create 3′ single-stranded tails.
One 3′ tail invades a homologous DNA duplex and pairs with complementary bases.
DNA polymerase extends the invading strand.
The displaced strand is used as a template to repair the second gap.
DNA ligase seals nicks → two Holliday junctions form.
🧠 Tip: “Cut, chew, invade, extend, seal — DSBR heals.”
What is the outcome of the DSBR mechanism?
Formation of two Holliday junctions, which can be resolved to produce either recombinant (crossover) or nonrecombinant (patch) DNA molecules, depending on cut orientation.
Why was the DSBR model initially controversial?
Double-strand breaks were thought to be lethal, but experiments showed they can stimulate homologous recombination and DNA repair, proving that DSBs can be repair intermediates, not dead ends.
🧠 Tip: “What kills can also heal — DSBs start recombination.”
How are recombination and replication connected in bacteria?
Recombination is essential to restart collapsed replication forks and initiate replication under DNA damage conditions — a process called Stable DNA Replication (SDR).
What is Stable DNA Replication (SDR)?
A form of replication initiation that occurs without new protein synthesis, triggered by DNA damage and double-strand breaks.
Does not require standard oriC initiation.
Uses recombination proteins to reload the replication machinery at DSBs.
Which proteins are required for Stable DNA Replication?
Pri proteins and DnaT help reload DnaB helicase via DnaC.
This mechanism allows replication to restart at DSBs, linking recombination to DNA replication restart.
🧠 Tip: “Pri + DnaT reload the fork when replication collapses.”
What is another major purpose of homologous recombination in E. coli besides evolution?
To facilitate DNA replication and repair, especially to restart collapsed replication forks.
Since bacterial chromosomes have only one origin of replication (oriC), a single nick or break can be fatal unless repaired by homologous recombination.
🧠 Tip: “One ori, one lifeline — recombination restarts the fork.”
What is the molecular basis of homologous recombination in E. coli?
Recombination proceeds through three coordinated steps:
DNA end processing → formation of single-stranded DNA (ssDNA) ends by RecBCD or RecQJ/RecFOR.
Synapsis formation → RecA binds ssDNA, promoting alignment and strand exchange with a homologous duplex to form a Holliday junction.
Resolution → RuvABC or RecG proteins resolve the Holliday junction to yield recombinant molecules.
🧠 Tip: “RecBCD cuts, RecA pairs, RuvABC repairs.”
What are chi (χ) sites, and why are they important?
Chi (χ) sites are short DNA sequence motifs (5′-GCTGGTGG-3′ in E. coli) that regulate RecBCD activity during recombination.
When RecBCD encounters a χ site, it reduces its nuclease activity and begins producing a 3′ single-stranded tail for RecA loading.
🧠 Tip: “χ = crossover hotspot; RecBCD shifts from destroyer to helper.”
What is the role of RecBCD in homologous recombination?
RecBCD is a helicase–nuclease complex that processes double-strand DNA ends.
It unwinds and degrades DNA until it reaches a χ site, then creates a 3′ ssDNA tail and loads RecA onto it to initiate recombination.
🧠 Tip: “RecBCD: chew, find χ, and hand DNA to RecA.”
Which enzymes resolve Holliday junctions in E. coli?
RuvA and RuvB mediate branch migration.
RuvC introduces targeted cuts to resolve the junction.
RecG can also promote junction migration and resolution in an alternate pathway.
🧠 Tip: “RuvABC resolves; RecG backs up.”
What are the main stages of homologous recombination in E. coli?
Generation of ssDNA ends → by RecBCD (from DSBs) or RecQJ/RecFOR (from gaps).
Synapse formation → RecA coats ssDNA and aligns it with a homologous duplex to form a Holliday junction.
Resolution → RuvABC or RecG proteins catalyze branch migration and resolution of the junction.
🧠 Tip: “RecBCD cuts, RecA pairs, Ruv/RecG repairs.”
What are χ (chi) sites, and what is their function during recombination?
χ (chi) sites are recombination hotspots (sequence: 5′-GCTGGTGG-3′).
When RecBCD encounters a χ site, it switches from DNA degradation to ssDNA generation and RecA loading, promoting homologous pairing.
🧠 Tip: “χ = signal to switch from chew to glue.”
What are the key recombination enzymes in E. coli and their functions?
RecBCD → processes DNA ends and loads RecA.
RecA → forms nucleoprotein filament, promotes strand invasion.
RuvA/B → catalyze branch migration.
RuvC/RecG → resolve Holliday junctions.
RecFOR/RecQJ → repair ssDNA gaps when RecBCD is not involved.
🧠 Tip: “RecBCD starts, RecA invades, Ruv/RecG finish.”
Why does E. coli have many recombination genes?
Because recombination is vital for DNA repair, genetic exchange, and replication restart, E. coli maintains multiple pathways and backup enzymes to ensure recombination can occur under various stress or damage conditions.
🧠 Tip: “One pathway fails? E. coli has backups to survive.”
What is RecBCD, and what role does it play in recombination?
RecBCD is a multi-enzyme complex that initiates homologous recombination by processing double-stranded DNA ends.
It unwinds DNA, degrades one strand, and produces a 3′ single-stranded tail for RecA loading at a χ site.
🧠 Tip: “RecBCD: unwind, chew, and hand off to RecA.”
What enzymatic activities are contained within the RecBCD complex?
RecBCD has:
ssDNA endonuclease activity → cuts single strands internally.
ssDNA exonuclease activity → digests DNA from free ends.
DNA helicase activity (two motors of opposite polarity).
DNA-dependent ATPase activity → powers unwinding and degradation.
🧠 Tip: “RecBCD = cut, chew, unwind, and fuel.”
Why is RecBCD critical for E. coli DNA repair and recombination?
It processes broken DNA ends to initiate strand invasion by RecA and to restart replication after fork collapse — linking DNA repair, recombination, and replication restart.
Without RecBCD, E. coli is highly sensitive to DNA damage.
🧠 Tip: “No RecBCD, no repair.
What happens when RecBCD reaches a χ (chi) site during recombination?
RecD is inactivated, halting excessive nuclease activity.
The complex shifts from DNA degradation to RecA loading on the new 3′ ssDNA tail.
🧠 Tip: “χ = switch signal — stop chewing, start recombining.”
How does RecBC stimulate RecA binding after χ site recognition?
RecBC promotes cooperative binding of RecA to the exposed 3′ ssDNA, forming a RecA–DNA filament capable of searching for homologous sequences and initiating strand invasion.
🧠 Tip: “RecBC hands DNA to RecA for pairing.”
Why is RecA loading tightly regulated by RecBCD?
Because Single-Stranded Binding (SSB) proteins bind ssDNA more strongly than RecA, random RecA binding could block normal replication or repair.
By loading RecA only after χ site recognition, RecBCD ensures that RecA binds exclusively at true recombination sites.
🧠 Tip: “RecBCD controls the gate—RecA loads only where it’s safe.”
What is the functional outcome of RecA loading by RecBC?
RecA polymerizes cooperatively on the 3′ ssDNA to form a helical nucleoprotein filament, the active substrate for homologous pairing and strand exchange.
This marks the commitment step of homologous recombination.
🧠 Tip: “RecA filament = green light for recombination.”
What is the sequence of the χ (chi) site in E. coli and which subunit recognizes it?
The χ site sequence is 5′–GCTGGTGG–3′, recognized by the RecC subunit of the RecBCD complex.
🧠 Tip: “C sees χ — C for chi recognition.”
What happens when RecC recognizes the χ site?
RecC interacts with the χ sequence and inhibits RecD, halting its strong helicase/nuclease activity.
RecD inhibition in turn reduces RecB’s 3′→5′ exonuclease activity, preventing further degradation of the DNA strand containing χ.
🧠 Tip: “C spots χ → shuts D down → saves the 3′ end.”
How do the RecB and RecD subunits modify their activity after χ recognition?
RecD (fast helicase) is inhibited.
RecB’s endonuclease is stimulated to cut the opposite (non-χ) strand across from the χ site.
RecB’s 5′→3′ exonuclease then degrades the non-χ strand, producing a 3′ ssDNA tail on the χ strand.
🧠 Tip: “After χ: D stops, B chops.”
How does RecB facilitate RecA loading after χ site processing?
Once the χ strand is exposed, RecB coats the 3′ ssDNA tail with RecA, forming a RecA–ssDNA filament that can invade homologous DNA and initiate recombination.
🧠 Tip: “RecB hands the baton to RecA at χ.”
What is the phenotype of recD⁻ mutants, and what does it tell us about χ sites?
recD⁻ mutants are viable, cannot degrade transformed linear DNA, but are still proficient for recombination.
This shows that χ sites are not strictly required for recombination—rather, they regulate RecBCD’s DNA-degrading activity.
🧠 Tip: “Lose RecD → less chew, still recombine.”
If χ sites aren’t essential for recombination, why does E. coli have them?
χ sites act as a defense mechanism — they provide “immunity” against foreign DNA.
χ sites are highly overrepresented in the E. coli genome (~1 per 4.5 kb, vs. 1 per 65 kb by chance).
DNA without χ sites (like foreign DNA or phage DNA) is more likely to be degraded by RecBCD.
🧠 Tip: “χ marks friend, not foe.”
How do phages overcome RecBCD-mediated degradation?
Phages protect their DNA by:
Shielding double-stranded ends, or
Producing proteins that inhibit RecBCD, such as the λ Gam protein.
🧠 Tip: “Gam jams RecBCD.”
Are χ-like sites found in other organisms?
Yes — many bacteria and archaea have χ-like sequences that interact with RecBCD-like enzymes.
These sequences differ between species but perform the same regulatory role in recombination and DNA protection.
🧠 Tip: “Different χ, same job — regulate recombination across life.”
What is a second reason E. coli maintains χ (chi) sites besides genome defense?
χ sites also control the direction of DNA replication following a double-strand break (DSB).
They ensure that any replication forks restarted by RecBCD proceed in the same direction as normal replication from oriC.
🧠 Tip: “χ keeps forks flowing forward.”
Why is the orientation (polarity) of χ sites important?
χ sites are polar—they only function when RecBCD approaches in the correct direction.
This ensures RecBCD produces a 3′ single-stranded tail on the correct DNA end for RecA loading and replication restart.
🧠 Tip: “Right way = recombine; wrong way = ignore.”
How are χ sites distributed in the E. coli genome, and why?
They are enriched in one orientation, biased so that RecBCD-mediated replication restart occurs co-directionally with replication from oriC.
This prevents head-on collisions between restarted and ongoing replication forks.
🧠 Tip: “Genome χ-biased — forks stay aligned.”
What is the RecF pathway, and how does it relate to RecBCD?
The RecF pathway is an alternate recombination route that generates single-stranded DNA ends for homologous recombination without RecBCD.
It acts mainly on gaps in DNA (not DSBs), using proteins like RecQ, RecJ, RecF, RecO, and RecR.
🧠 Tip: “RecF fixes gaps when RecBCD isn’t needed.”
What is the RecF pathway used for in E. coli recombination?
The RecF pathway initiates homologous recombination at single-stranded DNA (ssDNA) gaps created during:
DNA repair, or
Replication fork passage over a lesion in the lagging strand.
🧠 Tip: “RecF repairs gaps where replication stumbles.”
Why is the RecF pathway important?
It helps restart replication at stalled forks and ensures DNA repair continuity when the normal RecBCD double-strand break pathway isn’t available.
🧠 Tip: “When RecBCD rests, RecF revives the fork.”
What are the key proteins in the RecF pathway, and what do they do?
RecQ → a helicase that unwinds DNA near the gap.
RecJ → a 5′→3′ exonuclease that enlarges the gap to create ssDNA.
RecFOR complex → loads RecA onto the exposed ssDNA.
🧠 Tip: “Q unwinds, J chews, FOR loads RecA.”
What is the role of RecA once loaded onto ssDNA?
RecA binds ssDNA to form a helical nucleoprotein filament, the active structure for homologous pairing and strand invasion into duplex DNA.
🧠 Tip: “RecA filament = engine of recombination.”
How does RecA locate homologous sequences in the genome?
The RecA–ssDNA filament scans duplex DNA for complementary sequences, aligning them precisely to catalyze strand exchange and form a Holliday junction.
How efficient is RecA-mediated recombination?
Extremely efficient — ~100% of incoming ssDNA that encounters a complementary region in the cell recombines successfully, reflecting RecA’s strong pairing and catalytic activity.
🧠 Tip: “If it fits, it recombines — RecA almost never misses.”
How does the RecA nucleoprotein filament search for homology in DNA?
RecA-coated ssDNA scans duplex DNA along the major groove, testing for base-pair complementarity without disrupting the double helix.
🧠 Tip: “RecA reads the helix like Braille — through the groove.”
What is the next step after RecA identifies a complementary sequence?
The filament triggers displacement of one DNA strand, forming a D-loop (displacement loop) where the invading ssDNA pairs with its homologous strand.
🧠 Tip: “D-loop = Displaced strand loop — invasion confirmed.
What forms after the D-loop during strand invasion?
A triple-stranded structure forms temporarily, consisting of:
The invading ssDNA,
The paired complementary strand, and
The displaced original strand.
This is a critical intermediate in forming a Holliday junction.
Why is strand invasion by RecA considered remarkably precise?
Because RecA can align ssDNA and dsDNA to the exact base-pair level and initiate pairing without fully unwinding the duplex, preventing nonspecific recombination.
🧠 Tip: “RecA matches bases with surgical precision.”
What is trans-activation in the context of RecA-mediated recombination?
Trans-activation refers to the ability of a RecA–ssDNA filament to activate a non-complementary dsDNA molecule, allowing it to become temporarily open and receptive to invasion by another complementary strand.
🧠 Tip: “RecA primes one helix so another can invade.”
What might happen during trans-activation at the structural level?
The activated duplex may become transiently extended with partially separated strands, making room for the invading ssDNA to pair — a process still not fully understood mechanistically.
🧠 Tip: “RecA stretches the helix just enough for entry.”
What is spooling in RecA-mediated recombination?
After strand invasion, RecA can extend its nucleoprotein filament into neighboring dsDNA, effectively spooling the DNA through the filament.
This helps stabilize the invading strand and promote further pairing.
🧠 Tip: “RecA reels DNA in like thread on a spool.”
What special structures form after RecA-mediated strand invasion?
Junctions form where the invading ssDNA pairs with one strand of a dsDNA molecule — these are Holliday junction precursors, enabling crossover and branch migration.
🧠 Tip: “Invasion creates the crossover gateway.”
What is branch migration, and what does it accomplish?
Branch migration is the movement of the crossover point along the DNA duplex, extending or shifting the region of hybrid pairing (heteroduplex DNA).
It is catalyzed by specialized enzymes like RuvA, RuvB, and RecG.
🧠 Tip: “The branch walks — the heteroduplex grows.”
What are heteroduplexes in recombination?
Regions of DNA where complementary strands come from two different parental molecules and are base-paired together after branch migration.
🧠 Tip: “Hetero = mixed origin; duplex = double-stranded.”
Why are heteroduplexes important in recombination?
They represent the actual region of genetic exchange — mismatches here can lead to gene conversion or genetic variation after mismatch repair.
🧠 Tip: “Where hybrid pairing happens, new alleles are born.”
What is spooling in RecA-mediated recombination?
After strand invasion, RecA can extend its nucleoprotein filament into neighboring dsDNA, effectively spooling the DNA through the filament.
This helps stabilize the invading strand and promote further pairing.
🧠 Tip: “RecA reels DNA in like thread on a spool.”
What special structures form after RecA-mediated strand invasion?
Junctions form where the invading ssDNA pairs with one strand of a dsDNA molecule — these are Holliday junction precursors, enabling crossover and branch migration.
🧠 Tip: “Invasion creates the crossover gateway.”
Flashcard 3
What is branch migration, and what does it accomplish?
Branch migration is the movement of the crossover point along the DNA duplex, extending or shifting the region of hybrid pairing (heteroduplex DNA).
It is catalyzed by specialized enzymes like RuvA, RuvB, and RecG.
🧠 Tip: “The branch walks — the heteroduplex grows.”
What are heteroduplexes in recombination?
Regions of DNA where complementary strands come from two different parental molecules and are base-paired together after branch migration.
🧠 Tip: “Hetero = mixed origin; duplex = double-stranded.”
Why are heteroduplexes important in recombination?
They represent the actual region of genetic exchange — mismatches here can lead to gene conversion or genetic variation after mismatch repair.
🧠 Tip: “Where hybrid pairing happens, new alleles are born.”
What is isomerization of the Holliday junction?
Isomerization is a rearrangement of the four DNA strands within a Holliday junction that changes their crossover configuration.
It occurs rapidly, requires no energy, and does not break hydrogen bonds.
🧠 Tip: “The junction flips form without breaking bonds.”
Why is isomerization important for recombination outcomes?
The orientation of the junction at the moment of resolution determines whether the products are recombinant (crossover) or nonrecombinant (patch).
🧠 Tip: “How you cut decides who swaps.”
What is the structure and role of RuvA in recombination?
RuvA is a flower-shaped tetramer that binds Holliday junction DNA in a flat, square configuration.
It positions the four DNA arms for branch migration and cleavage.
🧠 Tip: “RuvA = anchor — holds the cross steady.”
What is the function of RuvB in the Ruv complex?
RuvB forms hexameric rings around opposite DNA arms.
It uses ATP hydrolysis to pump DNA through the junction, driving branch migration.
🧠 Tip: “RuvB = motor — moves the cross along.”
What does RuvC do during recombination?
RuvC acts as an endonuclease (resolvase) that cuts the Holliday junction at specific sequences (5′–(A/T)TT(G/C)–3′).
This resolves the junction into separate recombinant DNA molecules.
🧠 Tip: “RuvC = cutter — finishes the exchange.”
How do RuvA, RuvB, and RuvC function together?
They form the RuvABC complex:
RuvA binds and aligns the junction.
RuvB powers DNA movement.
RuvC resolves the junction by cutting.
Together they complete homologous recombination.
🧠 Tip: “A holds, B moves, C cuts — teamwork at the junction.”
What is the role of RecG in recombination and replication restart?
RecG can move Holliday junctions but cannot resolve them.
It can also bind three-stranded junctions, helping to reverse stalled replication forks at sites of DNA damage.
Pri proteins can then reload DnaB helicase to restart replication.
🧠 Tip: “RecG = the backup motor that saves stalled forks.”
How is recombination in E. coli different from that in higher organisms?
In E. coli, recombination usually occurs between a small linear DNA fragment and the bacterial chromosome, not between two large homologous chromosomes.
However, the enzymes and core mechanisms (RecA, RecBCD, Holliday junctions, etc.) are very similar to those in eukaryotes.
🧠 Tip: “Different scale, same machinery.”
Where do the linear DNA fragments involved in E. coli recombination come from?
They typically arise from:
Replication restart after fork collapse,
DNA damage, or
Incoming DNA (e.g., phage infection or transformation).
🧠 Tip: “Broken forks feed the recombination system.”
When does recombination between linear DNA and the E. coli genome occur?
It occurs during DNA replication, when homologous sequences are most accessible for strand invasion and pairing.
🧠 Tip: “Recombination piggybacks on replication.”
Outline the mechanistic steps for integrating a linear DNA fragment into the E. coli chromosome.
RecBCD processes the linear DNA end, producing a 3′ ssDNA tail at a χ site.
RecA coats the ssDNA and mediates strand invasion into the homologous chromosomal region.
Replication initiates from the invading DNA fragment.
Replication forks continue until reaching the terminus or a fork from oriC.
🧠 Tip: “Process → invade → replicate → merge.”
What happens to daughter cells after recombination between the chromosome and linear DNA?
After replication and segregation, each daughter cell can inherit different alleles if sequence differences existed between the incoming DNA and the original chromosome.
This produces genetic diversity in the bacterial population.
🧠 Tip: “Recombination = allele shuffle for evolution.”