Welcome to the mini-lecture on Gateway recombination, which is a form of site-specific recombination.
Unlike homologous recombination, which is a non-specific way to repair any broken DNA
end, site-specific recombination only occurs at very specific sites, making it a great
tool for molecular biology.
Shown here is a crystal structure of Integrase, the main enzyme in Gateway recombination,
forming a complex with its two target sites.
Notice that there are four protein molecules involved here – two in green and two in
purple – and the DNA strands are bent towards each other to allow for easy exchange.
As you will see, the integrase enzymes both cut and re-ligate the DNA while in this conformation.
By the end of this video, you should be able to: Compare and contrast the steps of PCR
cloning vs. Gateway cloning.
List the properties of the att sites.
Design a recombinase reaction by appropriately placing the att sites on substrate and target
DNA.
Explain the significance of negative selection with ccdB.
In PCR cloning, we use restriction enzymes to first create linear pieces of both our
gene of interest as well as the vector.
Then, the two linear pieces are re-joined using ligase, which will re-seal the DNA strands
into a circular molecule.
In contrast, Gateway recombination both begins and ends with circular DNA molecules.
The DNA backbones are both cut and re-ligated by the same enzyme while in complex with the
DNA, as seen in the crystal structure in the title slide.
These are the target and product sequences of the Gateway site-specific recombination
reaction.
As you look at the sequences, you may notice a few features.
First, these sequences all share a central nucleotide region, shown in black.
Second, unlike restriction enzyme sites, these sequences are NOT palindromic.
The non-palindromic sequence provides directionality.
Another property of these sequences is that they ALWAYS pair together: attB with attP,
and attL with attR.
L and R are named because they contain the LEFT and RIGHT arms of attP, respectively,
after cleavage and ligation takes place.
These are shown here in blue.
Different enzymes catalyze the forward and reverse reactions.
The commercial enzyme mix BP Clonase catalyzes the forward reaction of attB/attP into attL/attR.
This mix contains the integrase enzyme, whose activity will be described in the next few
slides, and the cofactor integration host factor, or IHF, a non-enzymatic protein that
binds DNA and assists in forming protein-DNA complexes.
These proteins are derived from viruses, which use these proteins to insert their viral DNA
into the genome.
The reverse reaction, which we will be performing, is catalyzed by the enzyme mix LR Clonase,
which includes the Excisionase, or Xis, protein in addition to integrase and IHF.
Xis is used by viruses to remove the viral DNA from the genome.
Xis interacts with Integrase and strongly drives the reaction in the opposite direction,
so each of these enzyme mixes are unidirectional.
One final feature is that integrase will make asymmetrical cuts in the common central region,
similar to a restriction enzyme, making sticky ends.
These will be re-annealed following strand swapping to generate the product sites.
We will now discuss the mechanism of the integrase enzyme in more detail.
The rate-limiting step in the recombination reaction is the formation of the protein-DNA
complex.
Each monomer, or single unit, of enzyme binds to one side of one recognition site, forming
a dimer on each att site.
The three-dimensional conformation of the dimers is different on the two different sites,
which allows them to properly pair.
The two dimers then come together, forming a complex with the two DNA strands in close
proximity.
The IHF cofactor assists in this process.
The formation of the protein-DNA complex is the first step in the recombinase reaction
cycle.
During the second step, a serine residue in the active site of each monomer attacks the
phosphate backbone of the DNA within the common central region.
This results in the creation of a sticky end of DNA (like in a restriction digest), as
well as a covalent bond between the protein and DNA backbone.
At this point, the three-dimensional conformation of the complex changes so that the strands
swap.
In the third and final step, the enzyme re-ligates the backbones together, similar to ligase.
The DNA is now no longer covalently attached to the enzyme, which is free to leave and
repeat the reaction at another site.
Note that after the strand swapping, there are different strands attached on either side
of the central region than before.
This results in different att sites at step 3 than step 1.
Let’s go through an example together.
In this case, the attB and attP sites are facing each other on either side of an orange
gene.
Remember that the attB and attP sites are always partners.
To make it easier to see the products of the reaction, and to mimic how the reaction works
in the test tube, let’s first re-draw the DNA so that the attB and attP sites are aligned.
Remember, these sites are directional, so they need to be lined up in parallel, with
the arrowheads facing the same direction.
At this point, the enzyme will cleave each site at the center, then switch which strand
is ligated to which backbone.
Again, these are directional, so the right-hand sides, or arrowheads, will always swap with
each other.
Finally, re-draw the product as a linear piece of DNA to see the results.
The attB and attP sites will ALWAYS result in attL and attR sites.
To identify which is which, remember that attL has the LEFT side of attP (shown here
in blue), and attR has the RIGHT, or arrowhead, side of attP.
The result on our orange gene in the middle is that it is now inverted.
You will have the chance to practice a few more of these in today’s activity.
Let’s do a quick concept check.
What will be the products of this reaction?
If you said “none,” you are correct!
The attL and attB sites will NEVER pair together!
So now, revisiting the Gateway reaction that YOU will be performing in lab, we can see
that our gene, CaMPARI, is surrounded by attL sites, and the destination vector has a region
surrounded by attR sites where our gene will go.
Using LR Clonase, the two regions will be swapped, creating attB and attP sites in our
new plasmids.
Note that unlike PCR cloning, there are never any linear intermediates.
In our system, we are using pDEST15 as our destination vector so that we can express
our protein in E. coli.
However, the advantage of the Gateway system is that the same entry vector could be used
to put our gene into a variety of other expression systems, without the need to design a new
cloning strategy!
There are also destination vectors for specific assays such as RNAi, two-hybrid, or antibody
production.
Another feature of this system is the negative selection of the plasmids that do not contain
our gene of interest.
Both the starting plasmid pDEST15 and the undesired ending plasmid, pENTR4 without CaMPARI,
contain the ccdB gene in place of our gene of interest.
Remember that this ccdB gene, shown in black, functions as a toxin, so any cells containing
it will not be able to grow following transformation.
This screening method greatly increases the probability of getting our desired product
plasmid.
This is the end of the presentation on site-specific recombination and Gateway cloning.
Hopefully now you are able to: Compare and contrast the steps of PCR cloning vs. Gateway
cloning.
List the properties of the att sites.
Design a recombinase reaction by appropriately placing the att sites on substrate and target
DNA.
Explain the significance of negative selection with ccdB.