Question 1

What are site-specific recombinases and how do they recognize DNA?

Accepted Answer

Site-specific recombinases are specialized enzymes that catalyze genetic exchange at defined DNA sites sharing sequence homology. These enzymes belong to Serine or Tyrosine recombinase families, distinguished by their active site residue. Recombinases bind to symmetric sequences flanking the regions to be exchanged, typically 20 to 30 base-pairs long, forming a synaptic complex before DNA cleavage and strand exchange occur.

Question 2

How do Serine and Tyrosine recombinases differ in their mechanisms?

Accepted Answer

Serine recombinases cut all DNA helices simultaneously before strand exchange, with the active site serine attacking the phosphodiester backbone at crossover sites. Tyrosine recombinases cut and join one DNA strand at a time, with the tyrosine residue covalently bonding to the 3' end of the cleaved strand. This creates a Holliday junction intermediate, which is resolved during the second crossover event to produce recombinant products.

Question 3

What are the three main outcomes of site-specific recombination events?

Accepted Answer

Integration occurs when circular DNA inserts into linear DNA. Excision happens when both sites are on the same molecule, removing a DNA section for integration elsewhere. Inversion results when incision sites are in opposite orientations, causing the DNA section to be removed and reintegrated in reverse orientation, as seen in Salmonella phase variation.

Question 4

What is phase variation and how does it relate to site-specific recombination?

Accepted Answer

Phase variation is a site-specific inversion process in Salmonella where a chromosomal segment inverts to produce two different flagellin protein types depending on environmental conditions. The inversion mechanism relies on site-specific recombination, allowing the bacterium to switch between flagellin variants by reversing the DNA orientation, enabling immune evasion and environmental adaptation.

Question 5

How are Cre and Flp recombinases used in genetic engineering?

Accepted Answer

Cre and Flp are tyrosine recombinases derived from bacteriophages used to mediate site-specific DNA insertions, deletions, and targeted protein expression in mammalian cells. Cre recognizes LoxP sites, which are 34 base-pairs long containing 13 bp palindromic sequences. Tissue-specific and ligand-inducible promoters provide spatial and temporal control over recombinase activity for precise genome editing.

Question 6

What is the main limitation of using site-specific recombination for genome editing?

Accepted Answer

The primary limitation is that recombination target sites must be pre-inserted or present naturally in the genome. Recent advances using mutagenesis and gene shuffling have designed Flp variants recognizing sites with combinatorial mutations, offering promise for creating more specific recombinase variants for commercial genome engineering applications.

Question 7

Why is site-specific recombination effective as a genetic engineering technique?

Accepted Answer

Site-specific recombination is efficient because DNA segments are cut and reorganized in a direction-specific manner, providing precise control over genetic modifications. This specificity enables targeted insertions, deletions, and inversions without random integration. The ability to use engineered recombinases with tissue-specific or inducible promoters allows researchers to control when and where recombination occurs in living organisms.

Ricombinazione conservativa sito-specifica e variazione di fase

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