Molecular Genetics

The Flp inversion system of the yeast 2 micron plasmid illustrates site-specific recombination ctalyzed by an Int-Flp recombinase.

Facts

The Flp inversion system was discovered on the 2 micron plasmid of S. cerevisiae and consists of a gene coding for a "recombinase" and two flanking "target" sites (located in the constriction in diagram). The Flp system functions to maintain the number of 2 micron plasmid copies at high levels in cells. Multiple copies result when inversion occurs after a replication fork has passed one of the target sites (note two forks travel in same direction and can not annihilate one another).

       #1       spacer       #2             #3        
 GAAGTTCCTATaC TTTCTAGA GAATAGGAACTTC C GAATAGGAACTTC
 CTTCAAGGATAtG AAAGATCT CTTATCCTTGAAG G CTTATCCTTGAAG

• A target site (above) has 3 sets of 13 bp repeats, an 8 bp and a 1 bp spacer. Inverted repeat pairs #1 and #2 are essential. The 8 bp spacer is the target for staggered endonucleolytic cuts.
• The spacer length is important, though the addition of 10bp is tolerated. The sequence of the spacer is not important as long as both sites are the same.
• If the spacer is palindromic, excision may result.
• The Flp recombinase is the only protein required for inversion. A covalent DNA-protein intermediate is formed through a tyrosine hydroxyl to a 3' phosphate. Both inter and intramolecular reactions are catalyzed. They are not specific as to direction.

Interpretations

• The Flp recombinase belongs to a different class of site-specific recombinases than the invertase-resolvase recombinases.
• In addition to the Flp-mediated inversion of one replication fork relative to the other, Flp should monomerize concatemers of the two micron plasmid that should result during this mode of replication.

Further information

• The Flp recombinase belongs to one of two classes of proteins that catalyze inversions. Other members of the family catalyze phage DNA integration. The Int-Flp family includes lambda Int, Flp, and cre.
• Three-dimensional structure studies provide insights into the mechanism used by the Flp recombinase to invert DNA sections.
• Cre is the only protein required to catalyze decatenation at lox target sites in processing replicated circles of bacteriophage P1. The lox spacer is 6 bp and 2 bp staggered cuts are made. The simplicity of the cre-lox system resulted in its adaptation in biotechnology to remove selectable marker genes from transgenic organisms to prepare them for commercial release. In another application, flanking lox sites can be used to exchange gene casettes between two DNAs.
• Lambda integrase requires several additional factors binding at sites surrounding the target "att" sites: xis, int, Hu and others.

This is page 3211 of Molecular Genetics by Ulrich Melcher, © 1997, 1998, 1999, 2010