Cleavage-dependent Ligation by the FLP Recombinase

Zhu, Xu-Dong; Sadowski, Paul D.

doi:10.1074/jbc.270.39.23044

Cited by 33 publications

(15 citation statements)

References 43 publications

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“…The collection of bands (Cl) above S (and therefore larger than S) indicate peptides linked to the labeled strand that could have arisen only from proteinase K action on the full-length Flp monomer. These results are consistent with previous observations that the phosphotyrosyl phosphodiester is a target for nucleophilic attack by Tyr-343 of Flp (1,23). Thus, formation of L in reactions with FS1 (lanes 2 and 3) can be accounted for by two modes of strand transfer: (i) by a single step reaction via the direct 5Ј-hydroxyl attack of the activated phosphate in FS1 (see also Fig.…”

Section: Resultssupporting

confidence: 81%

“…Consistent with earlier results, the activated substrates used here reinforce the role of base complementarity in bringing the 5Ј-hydroxyl in line with the phosphotyrosine bond (20). They also reveal that the phosphotyrosyl bond can be cleaved by Tyr-343 from a second Flp monomer bound across the spacer region (1,23). Whereas the strand joining reaction can occur in the activated substrates in the absence of, or following, cleavage by Flp, the alcoholysis reaction is absolutely dependent on the cleavage step.…”

Section: Discussionsupporting

confidence: 75%

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Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

Knudsen

Lee

Lisby

et al. 1998

Journal of Biological Chemistry

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The strand joining step of recombination mediated by the Flp site-specific recombinase involves the attack of a 3-phosphotyrosyl bond by a 5-hydroxyl group from DNA. The nucleophile in this reaction, the 5-OH, can be substituted by glycerol or other polyhydric alcohols. The strand joining and glycerolysis reactions are mechanistically equivalent and are competitive to each other. The target diester in strand joining can be a 3-phosphate covalently linked either to a short tyrosyl peptide or to the whole Flp protein via Tyr-343. By contrast, only the latter type of 3-phosphotyrosyl linkage is a substrate for glycerolysis. As a result, in activated DNA substrates (containing the scissile phosphate linked to a short The Flp site-specific recombinase of Saccharomyces cerevisiae is a member of the integrase family of recombinase proteins (2, 3). These proteins carry out recombination "conservatively," i.e. without DNA degradation or synthesis, and in the absence of exogenous energy input. This "break-join" mechanism follows from the ability of integrase-type recombinases to carry out two types of transesterification reactions, first to break the parental DNA strand and then to form the recombinant strand. The cleavage and joining steps of recombination follow a basic topoisomerase I strategy. Strand cleavage is mediated by an active site tyrosine residue, Tyr-343 in Flp. As a result, a 3Ј-phosphotyrosine bond and a 5Ј-hydroxyl group are formed at either side of the nick. Strand joining in the recombinant mode is chemically a reversal of the cleavage step, except that the reaction occurs between the broken strands of two DNA partners.Several interesting features of the cleavage and joining steps of the Flp reaction have been revealed (4 -7). The active Flp entity for strand cutting is a dimer (8, 9). Within the dimer, one Flp monomer, bound adjacent to a scissile phosphodiester bond, orients it for attack by Tyr-343 of the second Flp monomer bound across the strand exchange region (or spacer). Thus, the target phosphate is oriented in cis, while the cleavage nucleophile is donated in trans. The strand exchange reaction also follows the "cis-orientation/trans-nucleophilic attack" paradigm. In this case, the target phosphate belongs to the 3Ј-phosphotyrosine bridge, and the nucleophile is the 5Ј-hydroxyl group formed on the partner DNA. Once the target has been activated by Flp, a variety of exogenous nucleophiles can mimic the action of the native ones. Thus, strand breakage can be effected by tyramine, phenol, or hydrogen peroxide (6). Similarly, the 3Ј-phosphotyrosyl bond formed by Flp cleavage can be attacked by glycerol or polyhydric alcohols, by water (or the hydroxide ion), or even by a vicinyl 2Ј-hydroxyl group in a DNA-RNA hybrid substrate (10, 11).A simplified form of the recombination reaction (that involves the breakage and reformation of only one phosphodiester bond) can be performed using suitably designed "halfsite" substrates (12, 13). These substrates were fashioned after the half-sites employed for the...

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Section: Resultssupporting

confidence: 81%

Section: Discussionsupporting

confidence: 75%

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

Knudsen

Lee

Lisby

et al. 1998

Journal of Biological Chemistry

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“…Synthetic Substrates-The full-FRT and half-FRT sites were assembled by annealing complementary synthetic oligonucleotides in 5 mM MgCl 2 , 100 mM NaCl as described previously (27,35). The oligonucleotide containing 3Ј-phosphoryltyrosine was synthesized by R. Brousseau and C. Juby at the Biotechnology Research Institute, Montreal, Quebec, Canada, as described previously (36).…”

Section: Methodsmentioning

confidence: 99%

“…In Vitro Strand Exchange Assays-Strand exchange assays by Flp were done essentially as described by Zhu and Sadowski (27). DNA substrates (0.02 pmol) were incubated with Flp proteins (ϳ2.54 pmol) in 30 l of binding buffer containing 50 mM Tris-HCl (pH 7.5), 33 mM NaCl, 1 mM EDTA, and 2 g of sonicated and denatured calf thymus DNA at room temperature for 60 min by which time the ligation products had reached their maximal levels.…”

Section: Methodsmentioning

confidence: 99%

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The Role of Single-stranded DNA in Flp-mediated Strand Exchange

Zhu¹,

Sadowski

1998

Journal of Biological Chemistry

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The Flp recognition target site contains two inverted 13-base pair (bp) Flp binding sequences that surround an 8-bp core region. Flp recombinase has been shown to carry out strand ligation independently of its ability to execute strand cleavage. Using a synthetic activated DNA substrate bearing a 3-phosphotyrosine group, we have developed an assay to measure strand exchange by Flp proteins. We have shown that wild-type Flp protein was able to catalyze strand exchange using DNA substrates containing 8-bp duplex core sequences. Mutant Flp proteins that are defective in either DNA bending or DNA cleavage were also impaired in their abilities to carry out strand exchange. The inability of these mutant proteins to execute strand exchange could be overcome by providing a DNA substrate containing a singlestranded core sequence. This single-stranded core sequence could be as small as 3 nucleotides. Full activity of mutant Flp proteins in strand exchange was observed when both partner DNAs contained an 8-nucleotide single-stranded core region. Using suicide substrates, we showed that single-stranded DNA is also important for strand exchange reactions where Flp-mediated strand cleavage is required. These results suggest that the ability of Flp to induce DNA bending and strand cleavage may be crucial for strand exchange. We propose that both DNA bending and strand cleavage may be required to separate the strands of the core region and that single-stranded DNA in the core region might be an intermediate in Flp-mediated DNA recombination.The Flp gene of the 2-m circle plasmid of Saccharomyces cerevisiae encodes a conservative site-specific recombinase (Flp) that is involved in amplification of the plasmid (1-3). The Flp protein is a member of the integrase family of recombinases whose members share a tetrad of conserved residues (arginine 191, histidine 305, arginine 308, tyrosine 343; numbers represent amino acid numbers of Flp; see Refs. 4 and 5). The mechanism of Flp action is similar in many respects to that of the bacteriophage integrase and the Cre recombinase of bacteriophage P1 (6 -8).Flp mediates a recombination event between two 599-bp 1 inverted repeats. Embedded in each repeat are two Flp recognition target (FRT) sites that are the targets of action of Flp (Fig. 1). The FRT site contains two inverted 13-bp symmetry elements (a and b) which surround an 8-bp core region (open box). A third 13-bp symmetry element (c) is in direct orientation with element b but is dispensable for recombination both in vivo and in vitro (9 -11). Flp binds to each of the symmetry elements in an ordered fashion; binding to the b element occurs first, then binding to a, and finally binding to c (12, 13).Binding of an Flp molecule to a single symmetry element results in a bend of approximately 60°, whereas binding of Flp molecules to two symmetry elements (a and b) flanking the core region results in a severe bend of greater than 144°(referred to as a type II bend; see Refs. 14 and 15). Intermolecular protein-protein interactions between ...

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A new type of gene‐disruption cassette with a rescue gene for Pichia pastoris

Shibui

Hara

2017

Biotechnology Progress

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Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene-targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene-disruption system with a rescue gene using an inducible Cre/mutant-loxP system. With only short homology regions, the gene-disruption cassette of the system replaces its target-gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter-driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant-loxP sequences in the cassette is excised, leaving only the remaining mutant-loxP sequence in the genome, and consequently a target gene-disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color-change of the colonies. Then, the system was applied for knocking-out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene-targeting methods. All three gene-disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant-loxP system worked well to successfully isolate their knock-out mutants. This study identified a new gene-disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end-joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201-1208, 2017.

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Cleavage-dependent Ligation by the FLP Recombinase

Cited by 33 publications

References 43 publications

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

Alcoholysis and Strand Joining by the Flp Site-specific Recombinase

The Role of Single-stranded DNA in Flp-mediated Strand Exchange

A new type of gene‐disruption cassette with a rescue gene for Pichia pastoris

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