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Chapter 11 : Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae

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Abstract:

This chapter reviews the biochemical, mechanistic, and topological aspects of the Flp system with special emphasis on the contributions made by the Flp protein subunits and the target DNA to the individual steps of the recombination pathway. It illustrates how Flp recombination fits into the global physicochemical paradigm followed by the integrase/tyrosine family recombinases, while still retaining features that are strikingly different from other members of the family. The chapter then discusses how the Flp active site provides a model for the emergence of complex active sites from elementary active sites under the functional constraints faced by biological catalysts during their evolution. Next, it alludes to some of the applications of Flp in yeast and in extraneous host systems. Researchers conclude by considering potential mechanisms for Flp regulation in vivo to rapidly commission or decommission the amplification machine as demanded by the copy-number status of the 2μm plasmid. The DNA-protein interactions are mediated through both major and minor grooves. An impressive application of Flp in eukaryotes has been the creation of mosaic flies in by site-specific recombination between homologous chromosomes. Aside from its natural role in the physiology of the 2μm plasmid, Flp has been used to perform many artificial functions in its native host .

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11

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Figures

Image of Figure 1.
Figure 1.

The Flp reaction is initiated within a synaptic structure containing two DNA substrates and the four Flp monomers bound to them. Exchange of the first pair of strands results in a Holliday intermediate (H1). Isomerization of the complex (H2) promotes the exchange of the second pair of strands. This diagram anticipates several aspects of the Flp reaction that will be clarified in the text. For example, the two substrates are bent identically and arranged in an antiparallel orientation (L, left; R, right). The interactions between recombinase monomers bound to each substrate (the two darkly shaded monomers in substrate 1, and the two lightly shaded monomers in substrate 2) are responsible for the first strand cleavage and exchange reaction. The Holliday intermediate is roughly square planar, but strictly only 2-fold symmetric. During the resolution step, the catalytic dimers are formed between Flp monomers bound on the left and right arms of partner substrates. Each active dimer is constituted by a darkly shaded and a lightly shaded monomer. Note that, throughout the reaction pathway, a cyclic peptide connectivity between the four Flp monomers is maintained. The catalytically active and inactive associations between pairs of Flp monomers are indicated by the solid and dashed arcs, respectively.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 2.
Figure 2.

(A, left) A dimer of Flp bound to a full-site can assume one of two catalytic configurations. As a result, DNA cleavage can occur either at the left end of the spacer (top) or at the right end (bottom). In the cleaved complex, the Flp protein is linked to the 3′-phosphate end (p) by Tyr343, the cleavage nucleophile. (A, right) Analogous to the situation in the full-site, only half of the possible cleavages are observed within a dimer of a Flp-half-site complex ( ). A typical half-site contains one Flp-binding element, only two or three spacer nucleotides (n) on the strand containing the labile phosphodiester, and a full complement of the spacer nucleotides on the other strand ending in a 5′-hydroxyl group. This hydroxyl may be blocked by a phosphate group to prevent a cleavage reaction from continuing through the strand-joining step. During cleavage of the half-site, the short di- or trinucleotide product (5′OHnn3′) diffuses away and cannot take part in strand joining. For simplicity, the occupancy of the binding elements in the full-site and half-site by Flp is not shown. (B) In full-sites containing nucleotide bulges within the spacer on the top strand (left) or the bottom strand (right), strand cleavage is biased nearly exclusively to one end of the spacer (indicated by the short vertical arrows). The bulge-containing strand is the cleavage-susceptible one. Nucleotide bulges were created by the inclusion of three consecutive A’s on one strand with no complementary bases on the other. In the normal full-site (middle), cleavage can occur at either end of the spacer with roughly equal probability. The gel profile showing the Flp cleavage product(s) yielded by a substrate is shown below it. CL and CR stand for left and right cleavages, respectively. In the bulge-containing substrates, the cleavage bands are longer by 3 nt (because of the A3 bulge). (C) When the substrate has a fully mismatched spacer, both ends of the spacer may be cleaved. The 5′-hydroxyl groups at the cleaved spacer ends can take part in the indicated joining reactions to form hairpin products.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 3.
Figure 3.

The disposition of the active-site residues in Cre and human topoisomerase I (topo I) cocrystals with DNA in the cleaved and uncleaved states is shown. Hydrogen bonds are represented by dashed lines. In the Cre structures the RHRW tetrad and the tyrosine nucleophile are indicated ( ). For the topoisomerase I structure in the uncleaved state, the RKRH tetrad and the tyrosine nucleophile are depicted; however, in the cleaved form the histidine of the tetrad (His632) is disordered and is therefore not shown ( ). The uncleaved complex was obtained with topoI mutated at the active-site tyrosine (Y723F). In the structure shown, a tyrosine is represented occupying exactly the same position as the phenylalanine and is denoted as Phe723Tyr.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 4.
Figure 4.

Plausible roles of the active-site residues during the cleavage reaction by an integrase/tyrosine recombinase. The two arginines and the tyrosine nucleophile are represented around the DNA. The histidine, or histidine-tryptophan, pair of the RHRH/W tetrad could act as general base (B:) and general acid (BH), respectively, for deprotonating the tyrosine nucleophile and protonating the leaving group at its 5′ end. If the recombination mechanism is an adaptation of the classical pancreatic RNase mechanism, the roles of the two histidines or the histidine-tryptophan pair would be reversed during strand joining. The general base would now abstract the proton from the 5′-hydroxyl group, and the general acid would protonate the tyrosine-leaving group. Alternatively, if the role of the histidines is only to help stabilize the pentavalent intermediate, then the general acid and general base may simply be water.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 5.
Figure 5.

(A) The half-site substrate (S) containing the p form of the phosphorothioate at the scissile phosphodiester position (obtained by Klenow polymerase incorporation) should, in a two-step reaction, yield a hairpin recombinant (P) that retains the p configuration. The predicted diagnostic radioactive products of snake venom (SV) and P1 digestion are a dinucleotide and a mononucleotide, respectively, for both substrate and product. The experimental results shown at the right support this prediction. The mononucleotide band in the P1 digestion of the product indicates the level of phosphate contamination at the phosphorothioate position. The relative increase in this band in the P1 digestion of the substrate is caused by the second P position of the half-site. This labeled position is absent in the hairpin product. (B) The analysis is similar to that in A. The substrate containing the p form of the phosphorothioate at the cleavage position (preferentially cleaved by Flp over the p form) was prepared by ligating the tetranucleotide, 5′CpTTT3′, phosphorylated at the 5′ end by P to the appropriate half-site oligonucleotide. The mononucleotide band in the snake venom digestion is caused by phosphate contamination at the phosphorothioate position.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 6.
Figure 6.

(A) In the complementation reaction shown, the left half site (bold lines) is bound by FlpY343F and the right half site (thin lines) by a triad mutant of Flp. A functional active site (RHR, Y) assembled at the left half site cleaves the scissile phosphodiester. FlpY343F mediates strand joining within the cleaved intermediate to form the hairpin. The right half site is inactive in this reaction. (B) One good active site (RHR, Y) can be derived from FlpY343F and a single (I), double (II), or triple (III) triad mutant of Flp. The catalytically inactive states in I, II, and III are (F, HR), (F, R), and (F, ), respectively. The dimer formed by wild-type Flp and a triad, Tyr343 double mutant shown in IV, cannot yield a good active site (RH , Y in one case, and RHR, F in the other). (C) A half-site bound by FlpY343F can be cleaved by hydrogen peroxide or tyramine. The cleavage product from the tyramine reaction is active in the strand-joining step and yields the hairpin.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 7.
Figure 7.

(A) The potential modes of cleavage by a given Flp monomer were originally defined with respect to a parallel arrangement of the DNA substrates. The left and right DNA arms are indicated by L and R, respectively, with the subscripts referring to substrate 1 and 2. The cleavage modes numbered 1 through 4 represent horizontal, diagonal, and vertical, respectively. The curved arrows originating from the Flp monomer denote the delivery of Tyr343 nucleophile. (B) For an open-square arrangement of the DNA arms, which is close to what has been observed in Flp-bound Holliday junctions ( ), the cleavage modes numbered 2′ and 3 would be horizontal and diagonal, respectively.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 8.
Figure 8.

Two simultaneous strand cleavages can be mediated in a Y substrate by a Flp trimer. Subsequent strand joining yields the linear and hairpin recombinants (L and HP).

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 9.
Figure 9.

A free Holliday junction in the presence of divalent cations assumes the right-handed antiparallel stacked configuration (top left). When bound by Flp, the configuration switches to a nearly square planar form (right). In the context of recombination, the simplest scheme to produce this Holliday intermediate is to start with an antiparallel arrangement of the substrates (bottom left). The phosphodiester bonds involved in cleavage and exchange are indicated by the short vertical arrows.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 10.
Figure 10.

In the hybrid / plasmids, the accessory sites ( II/III) were used to build a topologically defined synapse, and recombination by Flp was conducted at the sites. In both the inversion and deletion substrates, the / sites were in direct repeat. The adjacent sites were in either inverted (A) or direct (B) orientation. The substrate plasmid was reacted with Flp either with (lane 3) or without (lane 2) resolvase present, then nicked with DNase I and electrophoresed. An unknotted product resulting from Flp inversion (A) would comigrate with the substrate. The number of knot nodes (3, 5, etc.) and catenane nodes (4, 6, etc.) in the recombination products are indicated. (C) A representation of a single hybrid / site. The protein monomers that associate with the binding sites are represented as circles: light grey for Flp, dark grey for Tn resolvase.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 11.
Figure 11.

Representations of Flp-mediated recombination adjacent to a 3-noded resolvase synapse. Recombination of direct-repeat sites proceeding through antiparallel site alignment requires an extra DNA crossing, and the product is a 4-noded catenane. Recombination of the inverted sites requires no additional crossing, and the product is a 3-noded knot. A possible pathway to the 4-noded catenane from a parallel synapsis of the direct sites is outlined in the table. However, this pathway would predict a 5-noded (rather than 3-noded) knot for the inverted sites. A and P indicate antiparallel and parallel alignment of sites.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 12.
Figure 12.

A recombinase dimer that cleaves in (I; Cre, for example) can, in principle, be related to one that cleaves in (Flp, for example) in one of two ways (II or III). The tyrosine (from the gray monomer) that cleaves at the left end of the spacer can be pushed to the right end in its functional orientation as shown in II. Alternatively, the tyrosine from the monomer at the right (white) can be moved to the left end as shown in III. The DNA configuration, indicated by the directed bend, is maintained the same in I, II, and III. The cleavage-susceptible phosphodiester positions are indicated by the black dots. Experimental evidence ( ) relates Cre and Flp by I and III, respectively.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 13.
Figure 13.

(A) Perspective of the cleaved Cre-DNA complex ( ) as viewed from a vantage point on the carboxyl-terminal side of the protein. Cleavage has occurred at the right-hand side, executed in the mode by the Cre monomer in white. (B) The same perspective as in the top panel is redrawn at the left to represent the helices K-N and their linkages. At the right, the change in connectivity between the gray and white monomers to mediate cleavage is shown. Effectively, the gray L helix is linked to the white M′-N′ helices. Conversely, the white L′ helix is linked to the gray M-N helices. The new location of the M-N helices approximates the situation for the recombinase tetramer seen in DNA-protein cocrystal structures.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 14.
Figure 14.

(A) In a cylindrical projection of the recombinase substrates with 6-, 7-, and 8-bp spacers as viewed along the helix axis, the scissile phosphodiesters at the near end are superimposed on each other (open circle). The relative angular displacements of those at the far end (filled circles) are indicated. (B) Recombinase monomers bound on L1/R1 and L2/R2 establish the included angle χ required for the first set of cleavages. The DNA dynamics associated with strand exchange (isomerization) establish χ between L1/R2 and L2/R1, promoting the second set of cleavages. The included angle gc; between two DNA arms represents the nonreactive configuration of the corresponding recombinase dimer.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 15.
Figure 15.

Isomerization between two alternative stacking patterns of the DNA arms within the Holliday junction can bring two scissile phosphodiesters into the proximal configuration, while simultaneously placing the other two in the distal configuration. The former pair is poised for cleavage and exchange, and the latter pair is in the nonreactive state. The functional outcome of this mode of isomerization is the same as that drawn in Fig. 1 . In both cases, isomerization switches the active recombinase pairs between the L1/R1 plus L2/R2 set on the one hand and the R2/L1 and R1/L2 set on the other.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 16.
Figure 16.

A half-site recombination mediated by Flp (A) is compared with Flp-RNase I (B) and Flp-RNase II (C). Flp RNase I, like the half-site reaction, is mediated by a “shared” active site and proceeds by Tyr343-mediated cleavage at the equivalent phosphodiester position. The only difference between the two is in the nucleophile used for the subsequent step: a 5′-hydroxyl group in panel A and a 2′-hydroxyl group in panel B. Flp-RNase II has a different target specificity and proceeds by a distinct mechanism that bypasses the tyrosine-mediated cleavage step.

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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Image of Figure 17.
Figure 17.

In a 2μm circle-derived hybrid plasmid, the Tn insertion is shown as a lollipop at the right. DNA segments drawn as parallel lines represent the 2μm circle and Tn repeats. A gene conversion event initiated by a Flp-induced break in one of the sites (the example shown here corresponds to cutting off the bottom site) can be terminated and resolved within the Tn repeats. The resulting plasmids contain a large inverted repeat, made up of the 2μm circle repeat, the Tn repeat, and the intervening gene-converted DNA. The plasmid products at the top and bottom are formed by the noncrossover and crossover modes of resolution, respectively. The parental and inverted configurations of Tn are indicated by the relative locations of the I restriction enzyme site (S).

Citation: Jayaram M, Grainge I, Tribble G. 2002. Site-Specific Recombination by the Flp Protein of Saccharomyces cerevisiae, p 192-218. In Craig N, Craigie R, Gellert M, Lambowitz A (ed), Mobile DNA II. ASM Press, Washington, DC. doi: 10.1128/9781555817954.ch11
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