1. DNA Recombination

2. Involves the physical exchange of DNA sequences from one molecule DNA to another molecule.
Importance of DNA recombination:
- providing genetic variation (Genetic variation is crucial to allow organisms to evolve in response to a changing environment).
- replacing damaged DNA with an undamaged strand.
- regulation of gene expression.
Major aspect of homologous recombination
- breakage of DNA
- joining of DNA

3. Key steps of homologous recombination
Alignment of two homologous DNA molecules.
Introduction of breaks in the DNA.
- These breaks are further processed to generate regions of single-stranded DNA.
Formation of initial regions of base pairing between recombining DNA molecules.
- Single-stranded region of DNA originating from one parental molecule pairs with its complementary strand in the homologous duplex DNA molecule (strand invasion, generating heteroduplex DNA).
Then, two DNA molecules become connected by crossing DNA strands generating Holliday junction.
- When the junction moves, base pairs are broken in the parental DNA molecules while identical base pairs are formed in the recombination intermediate, branch migration.
Clevage of the Holliday junction.
- Two pairs of DNA strands in the Holliday junction are cut during resolution.

5. Special endonuclease that simultaneously cut both strands of the double helix, creating a complete break in the DNA molecule.
The 5’ ends at the break are chewed back by an exonuclease, creating a protruding single-stranded 3’ ends.
These single stranded then search for a homologous DNA helix with which to pair, leading to the formation of a Holliday junction.
Strand invasion generates a Holliday junction that can move along the DNA by branch migration (increased the length of DNA exchanged).
If the two DNA molecules are not identical, branch migration through these regions of sequence difference generates DNA duplexes carrying one or few sequence mismatches.
Recombination is completed when Holliday junction is resolved (two recombining DNA molecules are separated).

6. Resolution of Holliday junctions occurs in one of the two ways and therefore give rise to two distinct classes of DNA products.
Horizontal resolution: Patch products or noncrossover products (No reassortment of flanking genes).
Vertical resolution: Splice products or crossover products (Reassortment of flanking genes).

8. Conservative site-specific recombination (CSSR)
- recombination between two defined sequence elements.
Transpositional recombination (transposition)
- recombination between specific sequences and nonspecific DNA sites.
Recombinases
- recognize specific sequences where recombination will occur within a DNA molecule
- bring specific sites together to form a protein-DNA complex bridging the DNA sites.
- catalyzes the cleavage and rejoining of the DNA molecules either to invert a DNA segment or to move a segment to a new site.

10. Conservative Site-Specific Recombination
Key feature: segment of DNA that will be moved carries specific short sequence elements, recombination sites (~20bp), where DNA exchange occurs.
Recombination sites carry:
i) sequences specifically bound by the recombinases.
ii) sequences where DNA cleavage and rejoining occur.
CSSR generate three different types of DNA rearrangements:
i) insertion of a segment of DNA into a specific site.
ii) deletion of a DNA segment.
iii) inversion of a DNA segment.
Example: Integration of the bacteriophage λ genome into bacterial chromosome.

11. Three types of CSSR recombination
which is depending on the organization of the recombinase recognition sites on the DNA molecule or molecules that participate in recombination.

12. Structures involved in CSSR
Each recombination sites is organized as a pair of recombinase recognition sequences and positioned symmetrically.
The recognition sequences flank a central short asymmetric sequence, known as crossover region, where DNA cleavage and rejoining occurs.
The subunits of the recombinase bind these recognition sites and recombination occurs.

13. Two families of conservative site-specific recombinases:
i) serine recombinases
ii) tyrosine recombinases
Key feature: covalent-DNA intermediate is generated when the DNA is cleaved
Serine recombinases:
- Side chain of the active-site serine residue attacks and then becomes joined to the
- The serine recombinases cleave all for strands prior to strand exchange. These double-stranded DNA breaks in the parental DNA generate four double-stranded DNA segments.
Tyrosine recombinases:
- Side chain of active-site tyrosine residue attacks and then becomes joined to the DNA.
- In contrast to the serine recombinases, the tyrosine recombinases cleave and rejoin two DNA strands first, and only then cleave and rejoin the other two stands.

14. Transposition
Moves certain genetic elements from one DNA site to another.
Mobile genetic elements are called transposable elements or transposons.
Movement occurs through recombination between the DNA sequences at the very ends of the transposable element and a sequence in the DNA of the host cell.
Transposons show little sequence selectivity in their choice of insertion sites. Therefore, transposons can insert:
i) within genes → disrupting gene function.
ii) regulatory sequences of a gene → alter gene expression.
Transposons are important cause of mutations leading to genetic disease in humans.
The ability of transposons to insert so promiscuously in DNA led to their modification and use as mutagens and DNA-delivery vectors in experimental biology.

15. Trsnposition of a mobile genetic element to a new site in the host DNA
Involves excision of the transposon from the old DNA location and insertion to a new site.
Or, one copy of the transposon stays at the old location and another copy is inserted into the new DNA site.

16. Three classes of Transposable Elements
DNA transposons
Viral-like retrotransposons
- long terminal repeat (LTR) retrotransposons
Non-viral retrotransposons
- poly A retrotransposons.

17. DNA Transposons
Carry both DNA sequences that function as recombination sites and genes encoding proteins that participate in recombination.
Recombination sites are at two ends of the element and are organized as inverted-repeat sequences.
Recombinases responsible for transposition are called transposases (or integrases).
Autonomous transposons
- carry a pair of terminal inverted repeats and a tranposase gene.
- function independently
Non-autonomous transposons
- carry only the terminal inverted repeats.
- require the presence transposase encoded by autonomous transposons to enable transposition.

18. Viral-like Retrotransposons
Carry long inverted terminal repeat sequences (LTRs).
Retrotransposon encode two proteins: integrase (transposase) and reverse transcriptase.
Reverse transcriptase uses RNA template to synthesize DNA.

19. Non-viral retrotransposons
Do not have the terminal inverted repeats present in the other transposon classes.
The two ends of the element have distinct sequences, 5’UTR and 3’UTR which is followed by poly-A sequence.
Carry two genes, ORF1 (RNA-binding protein) and ORF2 (reverse transcriptase and endonuclease).
c. Non-viral retrotransposons

20. DNA transposition by a Cut-and-Paste Mechanism
Transposase bind to the terminal inverted repeats at the end of the transposon.
Transposase brings the two ends of the tranposon DNa together to generate a stable protein-DNA complex (synaptic complex or transpososome which is essential to coordinate DNA cleavage and joining reactions on the two ends of the transposon’s DNA).
Transposase cleaves DNA such that transposon sequence terminates with free 3’-OH groups at each end of the element’s DNA.
3’-OH ends of the transposon DNA attack the DNA phosphodiester bonds at the site of new insertion.
Gap repair by DNA polymerase.

21. DNA transposition by a Replicative Mechanism
Element DNA is duplicated during each round of transposition.
Mechanism:
i) Assembly of the transposase protein on the two ends of the transposon to generate a transpososome.
ii) DNA cleavage at the ends of the transposon DNA. Transposase introduces a nick into DNA at each of the junctions between the transposon sequence and the flanking host DNA.
iii) The 3’OH ends of transposon DNA are then joined to the target DNA site by the DNA strand transfer reaction whereas 5’ ends of the transposon sequence remain joined to the old flanking DNA.
iv) The 3’-OH end in the cleaved target DNA serves as a primer for DNA synthesis. Replication proceeds through the transposon sequence and stops at the second fork.

23. Mechanism of retroviral integration and transposition of virus-like retrotransposons.
Transcription of the retrotransposon DNA sequence into RNA by cellular RNA polymerase.
The RNA is then reverse-transcribed to generate double-stranded DNA molecule, cDNA.
Integrase assembles on the ends of the cDNA and then cleaves a few nucleotides off the 3’ end of each strand.
Integrase then catalyzes the insertion of cleaved 3’ ends into a DNA target site in the host cell genome using DNA strand transfer reaction.
DNA repair proteins fill the gaps at the target site generated during DNA strand transfer to complete recombination.

24. Non-retroviral retrotransposon move by reverse splicing mechanism
Cellular RNA polymerase initiates transcription of and integrated LINE sequence.
The resulting mRNA is translated to produce products of the two encoded ORFs that then bind to the 3’ end of their mRNA.
The protein-mRNA complex then binds to a T-rich site in the target DNA.
The proteins initiates cleavage in the target DN, leaving 3’-OH at the DNA end forming an RNA:DNA hybrid.
The 3’-OH end of the target DNA serves as a primer for reverse transcription of the element RNA to produce cDNA.
The final steps of transposition reaction include second-strand synthesis and DNA joining and repair to crate a newly inserted LINE element.