Recombinase

Recombinases are enzymes that catalyze genetic recombination . This leads to a cleavage and reconnection of DNA sections, which leads to genetic diversity and enables the repair of mutated DNA.

Homologous recombination

The homologous recombination was first in 1964 by Robin Holliday described. It is based on the pairing of extensive homologous sequences, is common in bacteria and yeast, but inefficient in mammalian cells. This is related to the complexity and size of higher genomes and limits the application possibilities of the process for the targeted genetic modification of this cell type.

Site-specific recombination


Figure: site-specific recombinases: excision and integration using the example of the recombinases Cre and Flp . The recognition site of Cre recombinase ( loxP subheading [locus of crossing over of P1 phage]) comprising 34 base pairs in two 13 base pair contact points and an 8 base pairs comprehensive spacer ( " spacer "). The Flp recognition site ( FRT [Flp-recombinase target]) is slightly longer due to an additional contact point (A) . If recombination occurs between two recognition sites in the same direction (symbol: half arrow), the flanked DNA segment is cut out as a circular molecule ( excision ). This process is extremely efficient due to the original spatial proximity of both detection points. For the systematic modification of higher genomes, the “ flox process ” (a DNA segment is flanked by two loxP sites and can therefore be removed at a definable point in time) has become extremely important (B) . The reverse reaction (integration of a circular vector ) is also possible in principle (B)* , but inefficient because* the vector must find the second recombination site (i.e. due to the size of the "search volume" in the nucleus). since it is cut out again immediately after recombination if the recombinase activity cannot be terminated. that the enzyme favors the excision. The “method of choice” for this purpose today is the RMCE cartridge exchange process , which bypasses these problems . The “flippase” reaction: a DNA segment flanked by two recombinase sites oriented in opposite directions is turned over (inversion; not shown).

Recombination events of this type proceed over short recognition sites such as those found in yeast and phage . Since it is possible to transfer the corresponding enzymatic apparatus into mammalian cells, an extremely efficient system is available for the genetic modification of even higher cells. This has been used increasingly for a number of years.

The most frequently used recombination systems of this class are derived from the recombinases Cre ( cyclization recombinase or causes recombination ) of the phage P1, Flp (named after the flippase activity by which yeast invert sequence segments ) or Xer . Both enzymes belong to the integrase family of recombinases, which currently comprises around 30 members. The following figure illustrates possibilities that result from these systems. In addition, the systematic mutagenesis of the recognition sites has enabled another type of reaction: the RMCE cassette exchange process , to which a separate entry is dedicated.

Medical importance

In 2007, German researchers succeeded in using a recombinase for HIV- 1 infected cells for the first time. Here, the viral DNA is separated from the genome of a CD4 helper cell with the help of a modified Cre recombinase ( Tre ). Whether the complete eradication of HIV-1 with the help of Tre recombinase will actually be possible in the future as a gene therapy treatment strategy must currently be assessed with great caution. There are a number of fundamental as well as technical problems that must be carefully analyzed and resolved in the coming years.

Nevertheless, the current research results allow at least theoretical thought games for complete eradication. If eradication is not possible, at least another, alternative approach to HAART could be given.

swell

^ B. Das, E. Martínez, C. Midonet, FX Barre: Integrative mobile elements exploiting Xer recombination. In: Trends in microbiology. Volume 21, number 1, January 2013, pp. 23-30, doi: 10.1016 / j.tim.2012.10.003 . PMID 23127381 .
↑ Sarkar, I. et al. (2007): HIV-1 proviral DNA excision using an evolved recombinase. In: Science. Vol. 316, pp. 1912-1915. PMID 17600219
↑ Frank Buchholz, Joachim Hauber: Tailor-made recombinase - a new glimmer of hope for HIV eradication. In: Retrovirus Bulletin. Vol. 2007, No. 3, pp. 9-11.

literature

Andrews, BJ et al. (1985) The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences. In: Cell. Vol. 40, pp. 795-803. PMID 3879971

Web links

DNA recombination: Holliday model
Site-Specific Recombination ( Memento from March 9, 2011 in the Internet Archive )

[1] B. Das, E. Martínez, C. Midonet, FX Barre: Integrative mobile elements exploiting Xer recombination. In: Trends in microbiology. Volume 21, number 1, January 2013, pp. 23-30, doi: 10.1016 / j.tim.2012.10.003 . PMID 23127381 .

[2] Sarkar, I. et al. (2007): HIV-1 proviral DNA excision using an evolved recombinase. In: Science. Vol. 316, pp. 1912-1915. PMID 17600219

[3] Frank Buchholz, Joachim Hauber: Tailor-made recombinase - a new glimmer of hope for HIV eradication. In: Retrovirus Bulletin. Vol. 2007, No. 3, pp. 9-11.