FLP site-specific recombinase of yeast 2-μm plasmid

Beatty, Linda G.; Babineau-Clary, Donna; Hogrefe, Christine; Sadowski, Paul D.

doi:10.1016/s0022-2836(86)80003-1

Cited by 29 publications

(18 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The topological products resulting from recombination of supercoiled substrates produced by the integrase or the Tn3 resolvase were found to have an invariant stereostructure: the nodes are positive in sign for the integrase and negative for the Tn3 resolvase (19 -21). Recombination of a relaxed excision substrate by the Flp recombinase produces only unlinked products rather than a mixture of linked and unlinked circles (22). This is consistent with recombination in a unique sense within a synapse of unique topology.…”

supporting

confidence: 68%

“…The reaction was preincubated at 30°C and was initiated by the addition of Flp. The reaction was terminated after 20 min, and the DNA was phenol-extracted, ethanol-precipitated, and suspended in a small volume of TE buffer as described by Beatty et al (22). The 20-min time was chosen because it was early in the reaction before the appearance of the site-specific topoisomerase activity of Flp that occurs late in the reaction (Ref.…”

Section: The Enzymesmentioning

confidence: 99%

“…However, if the two recombining FRT sites are located on the same supercoiled molecule, topological analysis can be used to distinguish two rounds of recombination from simple reversal of the reaction. Flp recombines supercoiled substrates by a mechanism of "random collision" (22). This means that during synapsis and subsequent recombination of the two FRT sites, random numbers of the interdomainal supercoils are trapped as nodes between the recombining sites (Ref.…”

mentioning

confidence: 99%

“…This means that during synapsis and subsequent recombination of the two FRT sites, random numbers of the interdomainal supercoils are trapped as nodes between the recombining sites (Ref. 22, Fig. 2).…”

mentioning

confidence: 99%

“…If the sense of the strand exchange by Flp is the same in each round (see above), when the products of the first round undergo a second round of recombination, the products are knots with odd numbers of nodes (for excision substrates) or knots with even numbers of nodes (for inversion substrates), respectively, and both are in a nonrecombinant configuration (Ref. 22, Fig. 2).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Topological Analysis of the Role of Homology in Flp-mediated Recombination

Azam

Dixon

Sadowski

1997

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Recombination by the Flp recombinase of Saccharomyces cerevisiae is known to be inhibited by heterology of the overlap regions of the two recombining DNA targets (FRT sites). We have used topological analysis to show that Flp can promote two rounds of intramolecular recombination between heterologous FRT sites contained within the same supercoiled plasmid. The products are in parental nonrecombinant configuration. Thus, heterology may appear to "block" recombination by rendering the heteroduplex recombinant products unstable, thus favoring a second round of recombination to homoduplex (but parental) products. Hence, homology in the core region is not a requirement for the recombination reaction by Flp but for the formation of recombinant products.Flp is a conservative site-specific recombinase that is a member of the integrase family (1). Flp is encoded by the 2-m circle, an autonomously replicating, multicopy plasmid of Saccharomyces cerevisiae (2), and plays a role in the amplification of the 2-m plasmid (3-5).The Flp recognition target (FRT) 1 sites lie within two 599-bp inverted repeats of the plasmid. An FRT site contains three 13-bp Flp binding sites, symmetry elements a, b, and c (Fig. 1). Two symmetry elements (b and c) lie in the same orientation, while symmetry element a lies in reverse orientation separated from symmetry element b by an 8-bp asymmetrical core. The deletion of symmetry element c has no detectable effect on recombination either in vivo or in vitro (6 -8).Flp cleaves the FRT site at the margins of the 8-bp core and becomes covalently attached to the 3Ј-phosphate via Tyr-343 within the active site (9 -11). Cleavage occurs by the cooperation of two Flp molecules and is said to occur in trans (12, 13). The Flp molecule that acts as the tyrosine donor is bound to a symmetry element across the core from the cleavage site (14). Two reciprocal strand exchanges generate a four-armed Holliday intermediate, which is then resolved by another pair of reciprocal strand exchanges to yield reciprocally recombinant products (15-18).A site-specific recombinase carries out strand exchange in a unique "sense." This means that for a given reaction, the direction of the strand exchanges (clockwise or counterclockwise) in relation to the helical axis of the paired DNA targets is always the same rather than random. The topological products resulting from recombination of supercoiled substrates produced by the integrase or the Tn3 resolvase were found to have an invariant stereostructure: the nodes are positive in sign for the integrase and negative for the Tn3 resolvase (19 -21). Recombination of a relaxed excision substrate by the Flp recombinase produces only unlinked products rather than a mixture of linked and unlinked circles (22). This is consistent with recombination in a unique sense within a synapse of unique topology. It could also be due to recombination in either sense within synapses of opposite sign.It has been observed that although the core sequence can be changed (23, 24), efficient recombinat...

show abstract

supporting

confidence: 68%

Section: The Enzymesmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations