Genetic Recombination and the Use of Recombinase in Cloning

I must admit that my friend and I spent part of the lecture playing Hangman, but hopefully the main details are fine :)

Be familiar with the ability of DNA ligase to join DNA molecules and how to overcome vector self-ligation.

Okay, you should be reasonably familiar with the idea that DNA ligase joins DNA molecules. If not, have another look at my post on enzymes involved in DNA replication and repair.

Now let's have a look at the idea of self-ligation. Self-ligation occurs when, instead of a fragment of DNA being inserted into a plasmid, the two cut ends of the plasmid simply snap back together (i.e. self-ligate). This can be easily prevented by using alkaline phosphatase to remove phosphate groups on the plasmids. Without these phosphate groups, the plasmid cannot self-ligate, but fragments can still be inserted. This does leave single-stranded nicks where the plasmid lacks phosphates, but the effect of this is negligible due to the size of the inserted fragment and the distance between the two nicks.

Be familiar with the types of site-specific recombination.

There are three main types of site-specific recombination: insertion, deletion or inversion. They're pretty much self-explanatory: insertion involves the insertion of a new DNA fragment, deletion involves its deletion, and inversion involves re-inserting it backwards (i.e. inverting it). These reactions are catalysed by recombinases, which are part of a larger family of enzymes known as integrases. There are two essential parts of the DNA itself that facilitate site-specific recombination: recognition sites for the recombinases, and a crossover region where cutting and rejoining occurs. The crossover region also confers directionality, which is important in the case of inversion.

Understand the mechanism of action of Cre recombinase.

Cre recombinase is a recombinase (duh) taken from Phage P1. Its original function is to circularise the phage genome when it infects bacteria. Cre recombinase recognises a certain site called the LoxP site. The LoxP site contains an 8 base pair core sequence which is not palindromic and therefore confers directionality. This 8 base pair core sequence is flanked by two palindromic 13 base pair sequences. (By palindromic, I mean that the one before the core sequence reads the same as the one after, but backwards.)

Cre recombinase is made up of four identical subunits, each of which has a tyrosine in the active site. This tyrosine breaks the linkage between the 5'-OH and the phosphate of a nucleotide, forming a covalent intermediate until another nucleotide comes along. There are other recombinases that have serine in the active site- these ones break the linkage between the 3'-OH and the phosphate of the next nucleotide. The formation of these intermediates eliminates the need for ATP or other energy sources.

Cre recombinase works by cleaving and rejoining two strands simultaneously. Subunits R1 and R2 hold onto one DNA molecule at the palindromic sequences while R3 and R4 hold onto the other (again, at the palindromic sequences).

One of the benefits of using Cre recombinase is that its recognition sequence, LoxP, doesn't occur naturally in plants or animals. Hence, engineering a LoxP site into the genome and then using Cre recombinase will not cause unwanted cuts. (Of course, there's probably a tiny chance that there could be the exact same site somewhere in the genome by chance, but that chance is really, really negligible at best.)

Be familiar with applications of Cre recombinase and LoxP sites in cloning and conditional gene targeting.

As I just mentioned, Cre recombinase and LoxP are good for "cutting and pasting" (similar to restriction enzymes) as LoxP sites don't occur naturally in most plants and animals. Hence they are good for modifying and moving around genes. LoxP sites can also be used for conditional gene targeting- the so-called "knocking out" of genes.

I'm now going attempt to explain conditional gene targeting, using mice as an example. To create mice that have a gene "knocked out," you first need to start with two kinds of transgenic mice. One has the gene for Cre recombinase tied to an inducible and tissue-specific promoter, whereas the other kind has loxP sites flanking one of the exons of the gene in question. Following breeding, some of the mice should have both characteristics: loxP sites and the gene for Cre recombinase. The gene that you are studying can then be "knocked out" by inducing the production of Cre recombinase. This allows more control over timing of gene expression, and also allows you to see the effects of genes that could have been lethal during embryonic development.