Recombination

Recombination

Ku70–Ku80 complex is involved in

Mammalian double-strand break repair choice depends on cell cycle and end processing factors. Ku70-Ku80 heterodimer is initial sensor avidly threading onto free DNA ends with sequence-independent high affinity, physically blocking extensive resection by MRN-CtIP and Exo1. By recruiting DNA-PKcs, XRCC4, Ligase IV, Ku drives classical non-homologous end joining pathway which re-ligates ends with minimal processing generating small indels. Loss of Ku unmasks ends to homologous recombination or alternative microhomology-mediated end joining, shifting repair balance toward resection-dependent mechanisms particularly active in S phase, illustrating competitive pathway control.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 12: Ku Complex Involvement in Classical NHEJ

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Recombination

Xis protein primarily promotes

Directionality of lambda recombination controlled by accessory proteins dictating which attachment sites synapse. Integration intasome formed by Int plus IHF favors attP x attB recombination generating attL and attR flanking prophage. For prophage escape after induction, Xis protein synthesized binds X binding sites in attR introducing bends that prevent Int from occupying P-type sites while favoring excisive contacts. Xis also contacts Fis and Int N-terminal domain redirecting tetramer towards attL x attR excisive mode. Thus Xis shifts equilibrium from integrative to excisive state promoting precise phage DNA excision and circularization.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 12: Xis Promotion of Lambda Excision

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Recombination

λ integrase requires which accessory protein for integration?

Lambda integrase integration pathway depends on elaborate nucleoprotein intasome rather than Int alone. Integration Host Factor heterodimer binds to attP arm regions inducing U-turn bending bringing distant Int binding sites P'1, P'2, H1 near core B and C sites. IHF bending reduces effective DNA persistence length, enabling cooperative interaction of Int monomers bridging attP and attB. Fis protein further modulates geometry. In absence of IHF, in vitro integration efficiency drops hundredfold. Therefore IHF serves as accessory factor essential for efficient prophage establishment, while excision additionally needs Xis for architectural remodeling.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 7: IHF Accessory Requirement for Lambda Integration

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Recombination

loxP sites in inverted orientation lead to

Outcome control by loxP orientation underpins Cre utility. Recombination between directly repeated loxP excises intervening segment as circle, often desirable for deletion. When repeats inverted, circular product cannot form because joining opposite strands flips orientation instead of deleting. After first strand exchange forming Holliday junction, resolution in inverted geometry rotates segment 180 degrees, reversing direction of coding sequences between sites. This inversion reaction is reversible as both resultant loxP sites remain in chromosome, allowing conditional invertible modules for gene expression toggling and lineage labeling relying on stable inversion instead of excision.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 8: Inverted loxP Sites Lead to Inversion

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Recombination

RuvC resolves Holliday junction by

Branch migration by RuvAB creates mobile junction but final separation into two duplexes demands nuclease action. RuvC is endonuclease that recognizes Holliday junction structure specifically, introducing paired nicks on opposing strands at preferred sequence 5'-(A/TTTG/C)-3' near crossover. Dimeric enzyme coordinates two active sites containing conserved aspartates binding magnesium, executing concerted cleavage producing ligatable products. Orientation of cuts determines outcome: north-south cutting yields non-crossover patches, east-west cutting yields crossover splices. RuvC action terminates recombination, resolving linkages that would otherwise block chromosome segregation during replication fork rescue and double-strand break repair.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 12: RuvC Cleavage Resolution of Holliday Junction

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Recombination

RuvB functions as

RuvB belongs to AAA+ ATPase superfamily associated with various cellular activities containing Walker A and Walker B motifs required for ATP binding and hydrolysis. It forms hexameric ring structures that encircle double-stranded DNA adjacent to RuvA-bound junction. ATP binding stabilizes oligomer on DNA, while hydrolysis drives rotation and translocation pulling DNA through RuvA scaffold. This motor activity powers ATP-dependent branch migration extending heteroduplex length without requiring strand cleavage. Mutation of Walker motifs eliminates ATPase activity and stalls recombination intermediate processing, demonstrating essential helicase-like function distinct from endonucleolytic resolution.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: RuvB as ATPase Helicase Motor for Branch Migration

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Recombination

RuvA protein binds to

Formation and processing of Holliday junction is organized by RuvAB complex. RuvA is tetrameric DNA-binding protein with preference for four-way junction architecture over linear duplex. It forms sandwich with two concave surfaces contacting crossover center, forcing junction into open square-planar configuration exposing two duplex arms for RuvB loading. RuvA thus acts as specificity factor delivering junction-processing motor to correct DNA structure and prevents off-target translocation on regular duplex. Acidic C-terminal domain of RuvA recruits RuvB hexamers via interaction with domain II, orchestrating downstream branch migration.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 10: RuvA Binding to Holliday Junction Structure

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Recombination

RecA-mediated homology search requires minimum homology of

Homology search by RecA family does not require long perfect match for initial pairing, but minimal stretch of stable base pairing is necessary to maintain joint molecule. Biophysical single-molecule studies reveal that invasion requires approximately 8 base-pair microhomology for initiation, while stable D-loop that resists helicase rejection requires roughly 15 base-pair contiguous homology sampled in triplets. Below threshold, heteroduplex dissociates rapidly. This requirement prevents promiscuous recombination between short repeats causing deletions, while permitting efficient pairing between sister chromatids or homologs sharing genome-wide homology, ensuring fidelity and suppressing ectopic recombination.

Ref: NCBI Bookshelf, Biochemistry, RecA Homology Search and Minimum Homology Requirement

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Recombination

RecA binds preferentially to

Single-stranded DNA preference of RecA ensures that recombinogenic ends not double-stranded regions are targeted. ssDNA generated after resection or RecBCD processing is immediately bound by SSB in bacteria or RPA in eukaryotes, which melts secondary structures. RecA mediator proteins such as RecFOR facilitate replacement of SSB with cooperative ATP-bound RecA nucleating into contiguous filament. Within filament ssDNA adopts extended conformation facilitating base-triplet scanning for homology on intact duplex. This selective binding avoids illegitimate association with undamaged chromosomes, channeling strand invasion specifically to broken resected termini requiring repair.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 12: RecA Preferential Binding to ssDNA

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Recombination

Chi site sequence in E. coli is

Physical mapping of Chi activity identified consensus octamer as recombination stimulator in Escherichia coli and related enteric bacteria. Sequence 5'-GCTGGTGG-3' is statistically overrepresented in E. coli genome particularly on leading strand core orientation consistent with break repair bias. Orientation-specific hotspot elevates recombination roughly eightfold in its vicinity downstream, but only when encountered from defined direction relative to RecBCD entry due to asymmetric recognition. This sequence is absent in unrelated organisms using different Chi; Bacillus AddAB recognizes unrelated pentamer, demonstrating species-specific adaptation linking Chi recognition to RecC structural diversity.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Chi Octamer GCTGGTGG and Recombination Hotspots

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Recombination

RecC subunit recognizes

Within RecBCD trimer, RecC subunit does not possess catalytic domains but functions as scanner for recombination hotspots. Crystal structures show tunnel where duplex DNA threaded before reaching helicase motors, containing specific pocket recognizing Chi octamer 5'-GCTGGTGG-3' in single-stranded form. Recognition occurs when RecBCD unwinds DNA, Chi strand exposed interacts with RecC residues. Binding triggers conformational rearrangement transmitted through flexible linker changing RecB and RecD motor speeds and attenuating nuclease, effectively transforming enzyme from destructive nuclease to RecA loading machinery, explaining RecC mutations abolishing Chi response without affecting helicase activity.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 10: RecC Recognition of Chi Site Mechanism

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Recombination

RecBCD complex has helicase and nuclease activities due to

RecBCD is 330 kDa tri-subunit complex acting as primary double-strand break processing machine in Escherichia coli. RecB contains N-terminal SF1 helicase and C-terminal nuclease domains, RecD provides second SF1 helicase of opposite polarity with 5'→3' movement, while RecC is catalytically inert recognition subunit with channel for Chi. Both RecB motor drives 3'→5' translocation and RecD drives 5'→3' translocation enabling rapid unwinding up to 1000 base pairs per second. Upon Chi recognition nuclease activity switched, generating 3' tail for RecA loading mediated via RecB domain contacting RecA.

Ref: NCBI Bookshelf, Molecular Cell Biology, RecBCD Helicase and Nuclease Activities of RecB RecD

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