The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site.

Senecoff, Julie F.; Brückner, R.; Cox, Michael M.

doi:10.1073/pnas.82.21.7270

Cited by 148 publications

(88 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A neomycin resistance gene under the control of the phosphoglycerate kinase promoter and flanked by frt sites (Possemato et al, 2002) was inserted in an intron to facilitate selection of integrated clones. The frt sites allowed removal of the neo cassette in ES cells by transient lipofection with a flp recombinase-expressing plasmid (Senecoff et al, 1985). ES cells with a conditional allele of Dnmt3a (Dnmt3a 2lox ) (Fig.…”

Section: Generation Of Conditional Dnmt3a Knockout Micementioning

confidence: 99%

Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan

Nguyen¹,

Meletis²,

Fu³

et al. 2007

Developmental Dynamics

176

134

View full text Add to dashboard Cite

DNA methylation is an epigenetic mechanism involved in gene regulation and implicated in the functioning of the nervous system. The de novo DNA methyltransferase Dnmt3a is expressed in neurons, but its specific role has not been clarified. Dnmt3a is activated around embryonic day 10.5 in mouse neuronal precursor cells and remains active in postmitotic neurons in the adult. We assessed the role of neuronal Dnmt3a by conditional gene targeting. Mice lacking functional Dnmt3a in the nervous system were born healthy, but degenerated in adulthood and died prematurely. Mutant mice were hypoactive, walked abnormally, and underperformed on tests of neuromuscular function and motor coordination. Loss of Dnmt3a also led to fewer motor neurons in the hypoglossal nucleus and more fragmented endplates in neuromuscular junctions of the diaphragm muscle. Our results implicate a role for Dnmt3a in the neuromuscular control of motor movement.

show abstract

Section: Generation Of Conditional Dnmt3a Knockout Micementioning

confidence: 99%

Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan

Nguyen¹,

Meletis²,

Fu³

et al. 2007

Developmental Dynamics

176

134

View full text Add to dashboard Cite

show abstract

“…1) (31)(32)(33). The alignment of two sites during recombination is also mediated by asymmetry in the spacer.…”

Section: Introductionmentioning

confidence: 99%

“…There are two 12-bp FLP protein binding sites within FRT (25)(26)(27)(28)(29)(30). These include the external base pair of the spacer on each side plus the proximal 11 (31)(32)(33). The alignment of two sites during recombination is also mediated by asymmetry in the spacer.…”

mentioning

confidence: 99%

FLP recombinase is an enzyme.

Gates

Cox²

1988

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

The FLP protein of the yeast 2-jum plasmid catalyzes intermolecular site-specific recombination with a turnover number of 0.12 min-(per FLP monomer) for relaxed DNA substrates. Under conditions that enhance its stability, the protein can be used in catalytic rather than stoichiometric amounts. The reaction rate exhibits a strong dependence on FLP protein concentration even when the protein is present in excess relative to available recombination sites.Site-specific recombination events play crucial roles in DNA transposition, partitioning of extrachromosomal elements, gene regulation, and generation of genetic diversity (1). A number of site-specific recombination systems have been characterized in vitro. These include the bacteriophage A integration system (2), the resolvases derived from the Tn3 class of bacterial transposons (3, 4), the bacterial invertases (5, 6), the Cre-lox system of bacteriophage P1 (7), and the FLP system of the 2-gm plasmid of Saccharomyces cerevisiae (8, 9).Studies of these systems have provided detailed information about the architecture of the recombination sites, protein-DNA interactions, juxtaposition of two recombination sites, and key reaction intermediates (1,(10)(11)(12)(13)(14). A potentially useful .approach to many questions that remain is the application of classical enzyme kinetic methods. No detailed kinetic description of a site-specific recombination reaction has appeared. A number of factors contribute to the lack of information about kinetic parameters in these systems. The assays are often tedious, instability of some recombinases may affect reproducibility, and the reactions themselves are complex. Perhaps most important is the fact that it is not clear that these proteins are enzymes. The "recombinase" in each system appears to be required in stoichiometric rather than catalytic amounts (1,3,(15)(16)(17). Enzymatic turnover has not been demonstrated in any case. The focus of this report is turnover in the yeast FLP recombination system.The yeast 2-,um plasmid encodes a site-specific recombination system (FLP) that inverts the two unique regions of the plasmid (18). This inversion event is central to the strategy by which the plasmid amplifies its copy number (19-21). The only protein required is the plasmid-encoded FLP protein (22), which has recently been purified to near homogeneity (23,24). The relatively small recombination site (FLP recombination target or FRT) has been characterized extensively. The FLP protein will promote inversions, deletions, or intermolecular recombination efficiently if appropriate substrates are provided. Supercoiled and relaxed substrates react efficiently in vitro.The minimal FRT site consists of two 13-base-pair (bp) repeats separated by an 8-bp spacer (Fig. 1) (31)(32)(33). The alignment of two sites during recombination is also mediated by asymmetry in the spacer. FRT sites with symmetrical spacers remain functional but permit either of the two possible alignments of two sites during recombination (32). Several of the prop...

show abstract

“…The FLP protein cannot cleave a substrate that has a 4-bp insertion in the core region although it binds to it with good efficiency (2). Recombination between FLP sites with 1-bp insertions or deletions in the core region and an unaltered site is reduced (20). However, if both sites carry the same core-size mutation, the efficiency of recombination is relatively unaffected.…”

Section: Resultsmentioning

confidence: 99%

“…Presumably, the observed binding is altered such that the enzyme is unable to cleave and initiate the recombination event. Less drastic alterations in the size of the core region (addition or deletion of 1 bp) allow recombination to proceed, albeit with reduced efficiency (20). To further define the importance of the core sequence in recombination, we examined the activity of a point mutation in the core region.…”

Section: Resultsmentioning

confidence: 99%

Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

Andrews¹,

McLeod²,

Broach³

et al. 1986

Mol. Cell. Biol.

View full text Add to dashboard Cite

The 2,um plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two 599-base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro. We analyzed the interaction of the purified FLP protein with the target sequences of two point mutants that exhibit impaired FLP-mediated recombination in vivo. One mutation lies in one of the 13-bp repeat elements that had been previously shown to be protected from DNase digestion by the FLP protein. This mutation dramatically reduces FLP-mediated recombination in vitro and appears to act by reducing the binding of FLP protein to its target sequence. The second mutation lies within the 8-bp core region of the FLP target sequence. The FLP protein introduces staggered nicks surrounding this 8-bp region, and these nicks are thought to define the sites of strand exchange. The mutation in the core region abolishes recombination with a wild-type site. However, recombination between two mutated sites is very efficient. This result suggests that proper base pairing between the two recombining sites is an important feature of FLP-mediated recombination.Analysis of site-specific recombination in procaryotes has revealed many details about the nature of the molecular interactions that take place at the site of recombination. Much of this research has involved comparative analysis of the protein-binding capability and recombination activity of intact and altered target sequences (1, 4, 7, 13). The behavior of mutant target sites in binding and recombination assays suggests that the recombination reaction involves not only protein-DNA contacts but also DNA-DNA and proteinprotein interactions (4). The existence of a plasmid-encoded recombinase in yeast has allowed this type of analysis to be undertaken in a eucaryotic system. To gain insight into the biochemical mechanism of recombination, we have been studying the FLP protein, encoded by the 2,im plasmid of Saccharomyces cerevisiae.The FLP protein promotes efficient recombination between two identical recombination sites in vivo (5) and in vitro (16,21). In yeast cells, recombination occurs efficiently across two precise 599-base-pair (bp) inverted repeat sequences on the 2,um circle, resulting in inversion of one unique region of the plasmid with respect to the other. In vitro, FLP promotes an analogous inversion reaction between two sites in inverse orientation and a deletion event when the two sites are in direct orientation (9,19,22). FLP also promotes intermolecular recombination in vivo and in vitro (9,12). DNase footprinting studies as well as deletion analyses indicate that the FLP site is contained within a region consisting of two 13-bp inverted repeat sequences (symmetry elements) and an 8-bp core region (2,8). Although DNase protection studies reveal that FLP interacts specifically with a third 13-bp symmetry element, recombination assays show that this repeat is not necessary for efficient recombination in vivo (12) or in vitro (2,8...

show abstract

The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site.

Cited by 148 publications

References 17 publications

Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan

Ablation of de novo DNA methyltransferase Dnmt3a in the nervous system leads to neuromuscular defects and shortened lifespan

FLP recombinase is an enzyme.

Interaction of the FLP recombinase of the Saccharomyces cerevisiae 2 micron plasmid with mutated target sequences.

Contact Info

Product

Resources

About