Download Site-specific recombination mechanisms exploit DNA

Biochemistry: Site-specific recombination mechanisms exploit DNA
topology
Through a series of elegant genetic experiments in the early eighties Piet van de
Putte (Laboratory of Molecular Genetics, Leiden University) determined that
bacteriophage (Mu) changes its host range through expression of different tail
fibers by changing the orientation of a specific DNA segment, the G segment, in
its genome1. The phage-encoded Gin recombinase protein specifically recombined
the G segment located between short inverted DNA sequences, but not between
the same sequences in direct repeat orientation. Since there were analogous sitespecific DNA recombination systems in transposons, that had the opposite
selectivity for DNA deletion, rather than inversion, the most competitive issue
driving the field was to figure out how this selectivity was achieved.
The puzzle was solved by ingenious experiments performed by Piet’s graduate
student Roland Kanaar in the late eighties in collaboration with Nick Cozzarelli, a
professor at UC Berkeley and an expert in DNA supercoiling. Roland combined
biochemical and electron microscopy experiments with a branch of mathematics
known as topology to devise how the Gin protein, together with a host-encoded
protein and an enhancer site on the DNA exploited supercoiling to achieve
selectivity for DNA inversion, rather than deletion2,3. At the Nucleic Acids study
group meetings in Lunteren Roland was well known for dazzling his audience with
a flexible ribbon model of DNA, which he was twirling around as a magician while
recombining the ribbons. Those who grasped topology could answer his question
as to which knotted or catenated product should come out of the operation he
just performed. The work still has general implications as it elucidated the
mechanism of how nucleoprotein complexes assembled at distant sites along a
DNA chain communicate with each other to provide selectivity during DNA
transactions.
1
2
3
Van de Putte et al., 1980, Nature 286, 218-222; PMID: 6250048
Kanaar et al., 1989, Cell 58, 147-159; PMID: 2546671
Kanaar et al., Cell 1990 62, 353-366; PMID: 2164890
The provided images show the Gin protein on supercoiled DNA bridging three
distant sites (top) and knotted DNA products of a reaction essential to solve the
mechanism using topology.
Source: Claire Wyman (EUMC)