CH14 – Site-Specific Recombination & Transposition

Site-Specific Recombination (SSR)

Definition & Essential Features

• Recombination reaction that occurs only at a short, pre-defined DNA sequence (recombination site).
• Typical site length: \approx 20-200\,\text{bp}.
• Two indispensable components:
  • DNA site(s): e.g. LOX P, FRT (Flp Recombination Target), HIX in Salmonella.
  • Site-specific recombinase (endonuclease/ligase in one): e.g. Cre (for LOX P), Flp (for FRT), Hin (for HIX).
• Net genetic outcomes dictated by relative orientation of the sites on the same DNA molecule:
  • Inverted repeats → inversion of intervening segment.
  • Direct repeats → excision/deletion of the intervening segment (can operate in reverse to mediate insertion).
  • Sites on separate molecules → integration (insertion) events.

Catalytic Chemistry

• Two recombinase families distinguished by active-site residue:
  1. Tyrosine recombinases (Cre, Flp)
  2. Serine recombinases (Hin, some phage integrases)
• Key mechanistic points:
  • Four recombinase monomers load: two per site (one per strand).
  • Active-site Tyr or Ser performs nucleophilic attack on phosphate → covalent 3′- or 5′-phospho-protein intermediate.
  • Strand exchange in two steps (first pair, then the other) → transient Holliday junction-like intermediate.
  • No ATP required; energy of cleaved phosphodiester bond conserved in protein–DNA intermediate.

Yeast 2-µm Plasmid Copy-Number Control (Flp/FRT)

• Two-micron plasmid harbors two FRT sites in opposite orientation flanking the origin of replication.
• When copy number drops, Flp recombinase is expressed.
• During bidirectional replication the two FRTs are replicated; Flp inverts the FRT-bounded segment mid-replication, reorienting one fork → both forks chase each other around the circle.
• Result: rolling replication that makes multiple plasmid copies after a single initiation event.

Phage P1 Lysogenic Maintenance (Cre/LOX P)

• Injected as linear DNA containing two LOX P sites.
• Cre recombinase circularizes the genome by recombining the sites.
• Unlike most lysogenic phage, circular P1 remains episomal (extra-chromosomal), genes repressed.
• Low copy number sensed → nick initiates rolling-circle replication, producing concatemer with LOX P units; Cre resolves into monomeric circles for packaging.

Biotechnology Applications of SSR

• Place recognition sites flanking a cassette → inducible excision, inversion, or insertion by timed expression of recombinase.
  1. Remove transcription terminator to activate a silent gene.
  2. Site-specific gene knock-in via recombination between chromosomal site and plasmid site.
  3. Allele exchange: two crossovers replace wild-type allele with mutant while placing wild-type on the excised plasmid.

Phase Variation in Salmonella enterica (Hin/HIX)

• Salmonella alternates between two flagellin genes (FljB vs FliC) to evade host immunity.
• Hin invertase binds two HIX inverted repeats within a supercoiled loop and flips a promoter-containing segment.
  • Orientation 1: promoter drives fljB + fljA (repressor) → FljA suppresses fliC.
  • Orientation 2: promoter flipped → fljB & fljA OFF, repression lifted → FliC expressed.
• Characteristics of Hin system:
  • Uses Ser active site.
  • Requires accessory DNA-bending protein FIS.
  • Only acts when both HIX sites are on the same supercoiled DNA domain.

Transposition

Minimal Transposable Element (TE)

• At least one gene: transposase (a recombinase).
• Flanked by terminal inverted repeats (IRs); sequence varies per TE, specifically recognized by its own transposase.

Three Mechanistic Classes

1. Conservative ("cut-and-paste")
   • TE excised from donor site and inserted into target; copy number unchanged.
2. Replicative
   • TE copied during transposition; donor retains original, target receives duplicate.
3. Retrotransposition (RNA-mediated)
   • TE transcribed → RNA.
   • Reverse-transcribed → cDNA → integration; always increases copy number.

Conservative / Cut-and-Paste Details

• Transposase forms synaptic complex on IRs.
• Double-strand breaks at both ends via hydrolysis → 3′-OH ends form hairpins.
• Transposase makes staggered cuts in target DNA; TE inserted, gaps filled by host polymerase.
• Staggering produces target site duplications (TSDs) (direct repeats) flanking inserted TE.

Replicative Pathway (“copy-and-paste”)

• Single-strand nicks at TE ends; TE joined to target creating Shapiro intermediate (donor & recipient fused).
• DNA replication across branches duplicates TE.
• Resolution of cointegrate (two TEs head-to-head) by homologous or site-specific recombination separates donor and target, each now bearing a copy.

Retrotransposition

• Two subclasses:
  1. LTR retrotransposons (Ty, Gypsy)
  • LTRs harbor promoters.
  • RNA transcribed by host \text{RNAP II}.
  • Reverse transcriptase (RT) plus integrase (IN) encoded within.
  • cDNA synthesis primed by a host tRNA ("extra-chromosomally primed").
  • IN inserts new copy with TSDs.
  1. Non-LTR retrotransposons (LINEs, SINEs)
  • Lack LTRs; often poly-A tail.
  • ORF encodes endonuclease/RT fusion.
  • Mechanism: target-primed reverse transcription (TPRT).
    • Endonuclease nicks target → 3′-OH primes reverse transcription of TE RNA directly at chromosomal site.
    • Second-strand cleavage & synthesis completes insertion.

Transposable Elements in Bacteria

• Mu acts both as a lytic/lysogenic phage and as a replicative transposon; insertion mutagenesis gives its name ("Mutator").

Transposons in Eukaryotes

• Roughly \approx 46\% of human genome = transposon-derived; \approx 90\% of those are retrotransposons.
• DNA TEs similar to bacterial ISs: e.g. Mariner family—encodes only transposase + IRs.
• Retrotransposon examples:
  • Ty (yeast) & Gypsy (Drosophila): LTR, extra-chromosomal priming.
  • LINEs (L1) & SINEs (Alu): non-LTR, TPRT.

Ty Retrotransposon Life Cycle (Saccharomyces)

1. Transcription from LTR promoter; RNA poly-adenylated.
2. Export to cytoplasm; translation of Gag, protease, RT, integrase.
3. Gag packages RNA & enzymes into virus-like particle (VLP).
4. Reverse transcription inside VLP → dsDNA.
5. VLP transports cDNA + integrase back to nucleus → integration with TSDs.

Retrotransposons vs. Retroviruses

• Retroviruses resemble LTR retrotransposons but carry an additional env gene coding for envelope/capsid proteins enabling extracellular infection.
• Shared gene architecture: gag–pol–(env) framed by LTRs.
• Replication: RNA genome reverse-transcribed, integrated, transcribed, packaged, and released by cell lysis or budding.

Evolutionary Interplay Between TEs & Hosts

Protein Homologies

• Many transposases & retroviral integrases share D–D–E catalytic triad → common evolutionary origin.
  \text{Asp}–\text{Asp}–\text{Glu} residues coordinate Mg^{2+} for DNA cleavage/strand transfer.

Life-cycle within a Host Genome

1. Horizontal entry (rare) introduces active, highly mobile TE.
2. Initial bursts of transposition may be deleterious (gene disruption).
3. Host and TE co-evolve: mutations accumulate in IRs or coding region → mobility attenuates or silences TE.

Co-option of TE Machinery — Immune System Example

• V(D)J recombination that generates antibody diversity parallels DNA transposition.
  • Recombination Signal Sequences (RSS) resemble IRs.
  • RAG-1/RAG-2 recombinases are evolutionarily related to transposases.
• Antibody light-chain locus architecture:
  • \sim 1{-}300 Variable (V) segments, 1{-}5 Joining (J) segments, one Constant (C).
  • RAG-mediated recombination picks one V and one J → transcription/translation yields unique variable domain.
  • Approximate potential diversity from V–J combinations: 300\times5 \approx 1.5\times10^{3} per light-chain; combinatorial pairing with heavy chains + junctional variability boosts total to \approx 10^{7} distinct antibodies.
• Hypothesis: an ancient transposon invasion supplied the RAG genes and RSS architecture, later domesticated for adaptive immunity.

Summary of Key Numerical Facts

• Recombination site length: 20-200\,\text{bp}.
• Human genome: \approx 46\% transposon-derived; 90\% of those are retrotransposons.
• Yeast 2-µm plasmid: amplification via Flp/FRT.
• Antibody repertoire: \approx 10^{7} specificities from <10^{3} germ-line segments.

Practical / Ethical / Evolutionary Implications

• SSR systems (Cre-LOX, Flp-FRT) are powerful genomic engineering tools—enable conditional knock-outs, gene switches, targeted insertions with minimal off-target effects when sites are unique.
• TE activity can drive genome evolution but can also cause disease (insertional mutagenesis, genome instability).
• Domestication of TE proteins (e.g. RAG recombinase, telomerase RT domain) illustrates how genomes repurpose mobile-element machinery.