Site specific recombination | How do site specific Recombinases work?

Summary

This video explains site-specific recombination, a process where DNA fragments are integrated into specific target sequences with high efficiency. The recombination process involves enzymes like serine and tyrosine recombinases, each with distinct mechanisms. Biological examples include Lambda phage integration in E. coli and Hin recombinase-mediated DNA inversion in Salmonella, which regulate flagellar gene expression. The video also highlights biomedical applications like Gateway cloning, a restriction-free cloning method, and the Cre-loxP system used for generating conditional mutants in mice, demonstrating the practical utility of site-specific recombination in research.

Takeaways

• 😀 Site-specific recombination involves precise DNA sequence targeting and orientation-dependent recombination events.
• 😀 Unlike general recombination, site-specific recombination requires specific homology and directional alignment of sequences.
• 😀 Key outcomes of site-specific recombination include DNA insertion, deletion, and inversion, all of which are orientation-dependent.
• 😀 Serine and tyrosine recombinases are the main enzymes involved in site-specific recombination, with differences in how they catalyze the process.
• 😀 Serine recombinases create DNA breaks, pass strands, and reseal them to form the recombined DNA sequence.
• 😀 Tyrosine recombinases work on one DNA strand at a time, creating and resolving breaks to form the recombination product.
• 😀 Lambda phage genomic integration into E. coli is a well-known example of site-specific recombination using integrase (a tyrosine recombinase).
• 😀 The Hin recombinase in Salmonella bacteria mediates DNA inversion, affecting flagella gene expression and resulting in alternation between two types of flagella.
• 😀 Site-specific recombination plays a key role in regulating gene expression, as shown in Salmonella's flagella switching mechanism.
• 😀 Biomedical applications of site-specific recombination include Gateway cloning, which uses recombinase enzymes instead of restriction digestion for cloning DNA sequences efficiently.
• 😀 The Cre-LoxP system is widely used in generating conditional mutants in mice, providing a method for precise gene manipulation through recombination at specific loci.

Q & A

What is site-specific recombination?

Site-specific recombination is a process where DNA fragments are integrated, deleted, or inverted at very specific locations on the DNA sequence, with precise orientation and homology requirements.

How does site-specific recombination differ from other types of recombination?

Unlike other recombination processes, site-specific recombination is highly directed, meaning the specific orientation of the recombination sites matters, resulting in targeted insertion, deletion, or inversion of DNA sequences.

What role do enzymes play in site-specific recombination?

Enzymes, specifically recombinases, are crucial for site-specific recombination. They catalyze the breaking and rejoining of DNA strands at specific sites, with different recombinases having either serine or tyrosine in their active sites.

What is the difference between serine and tyrosine recombinases?

Serine recombinases use a serine residue to attack the DNA backbone and perform recombination by breaking and rejoining both strands simultaneously. Tyrosine recombinases, on the other hand, break and exchange DNA strands one at a time, using a tyrosine residue in their active site.

Can you explain the mechanism of serine recombinase in site-specific recombination?

Serine recombinase forms a DNA-enzyme complex where the serine residue attacks the DNA backbone, creating a break in one strand. The strands are then passed and resealed, resulting in a recombined DNA sequence.

How does tyrosine recombinase work differently from serine recombinase?

Tyrosine recombinase works one strand at a time. It creates an enzyme-DNA complex, and the exchange of one strand occurs before the second strand is recombined. The process involves the breakage and rejoining of one strand at a time, unlike serine recombinase, which works on both strands simultaneously.

What is an example of biological site-specific recombination involving Lambda phage?

In the case of Lambda phage, the genome integrates into the E. coli genome via site-specific recombination at **attP** sites on the phage and **attB** sites on the bacterial genome. The enzyme integrase, a type of tyrosine recombinase, facilitates this process.

What role does Hin recombinase play in Salmonella flagella inversion?

Hin recombinase mediates the inversion of flagellar genes in Salmonella. The recombination process allows the bacterium to switch between H1 and H2 flagella by altering the orientation of flagellar gene expression, thus affecting its movement and immune evasion.

What is Gateway cloning, and how does it utilize site-specific recombination?

Gateway cloning is a technique that uses site-specific recombination to insert a gene of interest into a vector. This method bypasses the need for restriction enzymes and ligation, utilizing recombinase enzymes like BP Clonase to recombine **attP** and **attB** sites, facilitating efficient cloning.

How does the Cre-loxP system work in generating conditional mutants?

The Cre-loxP system involves the use of **Cre recombinase** to catalyze recombination between **loxP** sites flanking a target genomic region. This allows for conditional gene deletion or inversion, often used in creating tissue-specific or inducible mutants in organisms like mice.