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Chapter 2 : An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective

Authors: Makkuni Jayaram1, Chien-Hui Ma2, Aashiq H Kachroo3, Paul A Rowley4, Piotr Guga5, Hsui-Fang Fan6, Yuri Voziyanov7
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Affiliations: 1: Department of Molecular Biosciences, UT Austin, Austin, TX 78712; 2: Department of Molecular Biosciences, UT Austin, Austin, TX 78712; 3: Department of Molecular Biosciences, UT Austin, Austin, TX 78712; 4: Department of Molecular Biosciences, UT Austin, Austin, TX 78712; 5: Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Bio-organic Chemistry, Lodz, Poland; 6: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan; 7: School of Biosciences, Louisiana Tech University, Ruston, LA 71272
Content Type: Monograph
Publication Year: 2015

Category: Clinical Microbiology

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Abstract:

Tyrosine family site-specific recombinases (YRs), named after the active site tyrosine nucleophile they utilize for DNA strand breakage, are widely distributed among prokaryotes. They were thought to be nearly absent among eukaryotes, the budding yeast lineage () being an exception in that a subset of its members houses nuclear plasmids that code for YRs ( ). However, YR-harboring DIRS and PAT families of retrotransposons and presumed DNA transposons classified as Cryptons have now been identified in a large number of eukaryotes ( ). The presence of functional YRs encoded in Archaeal genomes has been established by a combination of comparative genomics and modeling complemented by biochemical and structural analyses ( ). Over 1300 YR sequences mined from bacterial genome databases have been organized into families and subfamilies, providing a better understanding of the evolutionary relationships among them ( ). These classifications also encourage investigations into the potential functional significance of YRs whose genes are present as pairs or trios in bacterial and plasmid genomes.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective
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Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
/content/10.1128/9781555819217.chap2.fig2-1
Figure 1
Tyrosine family site-specific recombination. (A) The two target sites, each bound by two recombinase monomers across the strand exchange region, are arranged within the recombination synapse in an almost perfectly planar, antiparallel fashion. The left and right arms of the sites are marked as L1, L2 and R1, R2, respectively. The reaction proceeds by the cleavage/exchange of one pair of strands to form a Holliday junction intermediate, isomerization of the junction, and exchange of the second pair of strands to give the recombinant products (L1R1 + L2R2 → L1R2 + L2R1). The scissile phosphates engaged by the “active” active sites at distinct stages of the reaction are indicated by the filled circles. (B) The “half-of-the-sites” activity, responsible for the two-step strand exchange mechanism, is revealed by the crystal structure of the Flp-DNA complex ( ). Within each recombination partner (left), the green Flp monomer (bound at R1 or R2) is poised to promote the cleavage of the scissile phosphate adjacent to it (red circle). The tyrosine nucleophile for cleavage is donated in by the neighboring Flp monomer (bound at L1 or L2; magenta). Following isomerization of the Holliday junction intermediate (right), there is a switch between the active and inactive Flp pairs, signifying the imminent cleavage of the scissile phosphates adjacent to Flp monomers bound at L1 and L2. The tyrosine nucleophiles are donated across DNA partners, in the R1 to L2 and R2 to L1 configuration.
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Figure 1

Tyrosine family site-specific recombination. (A) The two target sites, each bound by two recombinase monomers across the strand exchange region, are arranged within the recombination synapse in an almost perfectly planar, antiparallel fashion. The left and right arms of the sites are marked as L1, L2 and R1, R2, respectively. The reaction proceeds by the cleavage/exchange of one pair of strands to form a Holliday junction intermediate, isomerization of the junction, and exchange of the second pair of strands to give the recombinant products (L1R1 + L2R2 → L1R2 + L2R1). The scissile phosphates engaged by the “active” active sites at distinct stages of the reaction are indicated by the filled circles. (B) The “half-of-the-sites” activity, responsible for the two-step strand exchange mechanism, is revealed by the crystal structure of the Flp-DNA complex ( ). Within each recombination partner (left), the green Flp monomer (bound at R1 or R2) is poised to promote the cleavage of the scissile phosphate adjacent to it (red circle). The tyrosine nucleophile for cleavage is donated in by the neighboring Flp monomer (bound at L1 or L2; magenta). Following isomerization of the Holliday junction intermediate (right), there is a switch between the active and inactive Flp pairs, signifying the imminent cleavage of the scissile phosphates adjacent to Flp monomers bound at L1 and L2. The tyrosine nucleophiles are donated across DNA partners, in the R1 to L2 and R2 to L1 configuration.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 2
Organization of conserved catalytic residues within the recombinase active site. The arrangements of the catalytic hexad (Arg-Asp/Glu-Lys-His-Arg-His/Trp) and the tyrosine nucleophile in Cre, Flp and λ Int active sites are shown ( ). The states of the active site with the scissile phosphate uncleaved and cleaved are shown at the left and right, respectively. The role of the conserved Asp/Glu of the hexad in transition state stabilization is likely indirect, by promoting the structural integrity of the active site.
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Figure 2

Organization of conserved catalytic residues within the recombinase active site. The arrangements of the catalytic hexad (Arg-Asp/Glu-Lys-His-Arg-His/Trp) and the tyrosine nucleophile in Cre, Flp and λ Int active sites are shown ( ). The states of the active site with the scissile phosphate uncleaved and cleaved are shown at the left and right, respectively. The role of the conserved Asp/Glu of the hexad in transition state stabilization is likely indirect, by promoting the structural integrity of the active site.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 3
The assembly of the Flp active site in . (A) In the shared active site of Flp, the tyrosine nucleophile (Tyr-343) is delivered by the donor Flp as part of the M helix (shown in magenta) to the proactive site, whose residues are shown in green, of the recipient Flp. The van der Waals’ contacts made by Trp-330 (recipient Flp) with Ser-336 and Ala-339 (donor Flp) are important for the positioning of Tyr-343 (donor) ( ). The stacking of His-309 (recipient) over Tyr-343 is stabilized by His-305 (recipient) and His-345 (donor). (B) Consistent with the importance of Trp-330–Ala-339 interaction, the loss of active site function resulting from the W330A substitution can be rescued by the second site A339M mutation, which increases the side chain length at this position ( ). The red circle in A and B denotes the scissile phosphate.
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Figure 3

The assembly of the Flp active site in . (A) In the shared active site of Flp, the tyrosine nucleophile (Tyr-343) is delivered by the donor Flp as part of the M helix (shown in magenta) to the proactive site, whose residues are shown in green, of the recipient Flp. The van der Waals’ contacts made by Trp-330 (recipient Flp) with Ser-336 and Ala-339 (donor Flp) are important for the positioning of Tyr-343 (donor) ( ). The stacking of His-309 (recipient) over Tyr-343 is stabilized by His-305 (recipient) and His-345 (donor). (B) Consistent with the importance of Trp-330–Ala-339 interaction, the loss of active site function resulting from the W330A substitution can be rescued by the second site A339M mutation, which increases the side chain length at this position ( ). The red circle in A and B denotes the scissile phosphate.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 4
Challenges to the homology rule during tyrosine site-specific recombination. (A) The integrase (IntDOT) of the conjugative transposon CTnDOT catalyzes exchange of both strands between target sites that contain five consecutive nonhomologous positions within the 7 bp segments swapped between them ( ). (B) The folded form of the “+” strand of the CTXϕ phage contains an imposter target site for XerCD recombinase of its host bacterium, . Single-strand exchange mediated by the XerC active site between the phage DNA and the bacterial chromosome results in phage integration ( ). The heteroduplex integrant in (A) and the pseudo-Holliday junction in (B) are likely resolved via replication. The flat horizontal arrows indicate recombinase binding sites. The short vertical arrows denote points of strand cleavage.
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Figure 4

Challenges to the homology rule during tyrosine site-specific recombination. (A) The integrase (IntDOT) of the conjugative transposon CTnDOT catalyzes exchange of both strands between target sites that contain five consecutive nonhomologous positions within the 7 bp segments swapped between them ( ). (B) The folded form of the “+” strand of the CTXϕ phage contains an imposter target site for XerCD recombinase of its host bacterium, . Single-strand exchange mediated by the XerC active site between the phage DNA and the bacterial chromosome results in phage integration ( ). The heteroduplex integrant in (A) and the pseudo-Holliday junction in (B) are likely resolved via replication. The flat horizontal arrows indicate recombinase binding sites. The short vertical arrows denote points of strand cleavage.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 5
Flp-mediated knotting of supercoiled plasmids by recombination between two sites harboring nonhomology within the strand exchange region. (A) The first recombination event between two head-to-head (inverted) sites from a synapse containing an odd number of interdomainal (blue × red) supercoil crossings will generate a torus knot with the same number of crossings. The product from a synapse with one blue × red crossing will be an unknotted inversion circle, as it takes a minimum of three crossings to form the simplest knot. In the example shown, a 3-noded knot is formed from a 3-crossing synapse. A second recombination event after dissociation of the first synapse, and the assembly of a synapse, can give rise to a twist knot with four crossings. (B) For sites in head-to-tail (direct) orientation, the first recombination event from a synapse with an even number of interdomainal crossings yields a catenane with the same number of crossings. The product from a synapse with no crossings will be two unlinked deletion circles. The diagram illustrates the formation of a 4-noded catenane from a 4-crossing synapse. A second round of dissociative recombination can convert the 4-noded catenane into a 5-noded knot. In the reactions shown in (A) and (B), intradomainal supercoils (blue × blue or red × red crossings) are omitted for clarity, as they do not contribute to knot or catenane crossings. The products from the second rounds of recombination revert to the parental configuration. The noncomplementarity in the product formed by recombination between sites containing nonhomology in their strand exchange regions encourages a second recombination event that restores base pairing and parental DNA configuration ( ).
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Figure 5

Flp-mediated knotting of supercoiled plasmids by recombination between two sites harboring nonhomology within the strand exchange region. (A) The first recombination event between two head-to-head (inverted) sites from a synapse containing an odd number of interdomainal (blue × red) supercoil crossings will generate a torus knot with the same number of crossings. The product from a synapse with one blue × red crossing will be an unknotted inversion circle, as it takes a minimum of three crossings to form the simplest knot. In the example shown, a 3-noded knot is formed from a 3-crossing synapse. A second recombination event after dissociation of the first synapse, and the assembly of a synapse, can give rise to a twist knot with four crossings. (B) For sites in head-to-tail (direct) orientation, the first recombination event from a synapse with an even number of interdomainal crossings yields a catenane with the same number of crossings. The product from a synapse with no crossings will be two unlinked deletion circles. The diagram illustrates the formation of a 4-noded catenane from a 4-crossing synapse. A second round of dissociative recombination can convert the 4-noded catenane into a 5-noded knot. In the reactions shown in (A) and (B), intradomainal supercoils (blue × blue or red × red crossings) are omitted for clarity, as they do not contribute to knot or catenane crossings. The products from the second rounds of recombination revert to the parental configuration. The noncomplementarity in the product formed by recombination between sites containing nonhomology in their strand exchange regions encourages a second recombination event that restores base pairing and parental DNA configuration ( ).

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 6
Reactions of half-sites containing methylphosphonate substitution at the scissile phosphate position. (A) The structures of methylphosphonate (MeP) are compared to that of the native phosphate in DNA. There are two possible stereoisomers of MeP ( or ). (B) The possible reactions of a half-site containing MeP at the scissile phosphate position are illustrated. The 5′-hydroxyl group on the bottom strand of the half-site is blocked by phosphorylation to prevent it from taking part in a pseudo-joining reaction. Attack of the MeP bond by the active site tyrosine will give the MeP-tyrosyl intermediate, which may undergo slow hydrolysis. The hydrolysis product may also be formed by direct water attack on the MeP bond. The two-step (type I) and single-step (type II) reaction pathways are mechanistically analogous to the type I and type II RNA cleavage activities of Flp (see text).
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Figure 6

Reactions of half-sites containing methylphosphonate substitution at the scissile phosphate position. (A) The structures of methylphosphonate (MeP) are compared to that of the native phosphate in DNA. There are two possible stereoisomers of MeP ( or ). (B) The possible reactions of a half-site containing MeP at the scissile phosphate position are illustrated. The 5′-hydroxyl group on the bottom strand of the half-site is blocked by phosphorylation to prevent it from taking part in a pseudo-joining reaction. Attack of the MeP bond by the active site tyrosine will give the MeP-tyrosyl intermediate, which may undergo slow hydrolysis. The hydrolysis product may also be formed by direct water attack on the MeP bond. The two-step (type I) and single-step (type II) reaction pathways are mechanistically analogous to the type I and type II RNA cleavage activities of Flp (see text).

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 7
Distinct activities of Flp(R191A) and Flp(R308A) on an MeP-half-site. (A) Flp(R191A) cleaves the MeP-half-site (S) using Tyr-343 to form the protein–DNA adduct (revealed by SDS-PAGE; top) ( ). This intermediate is converted to the hydrolysis product (HP) (revealed by denaturing PAGE; bottom) in a subsequent slow reaction. Flp(R308A), by contrast, yields the hydrolysis product directly, without going through the MeP-tyrosyl intermediate ( ). (B) The binding of an Flp monomer to activates the scissile phosphate, leaving it exposed until the binding of a second Flp monomer delivers Tyr-343 to the active site in . (C) Concomitant with the binding of a Cre monomer to the LoxP site, Tyr-324 engages the scissile phosphate in , thus protecting it against direct water attack. (D) As vaccinia topoisomerase, like Cre, assembles its active site in , the scissile phosphate is protected at the strand cleavage step during DNA relaxation. However, the protein’s grip on DNA is loosened during the strand rotation step, leaving the phosphotyrosyl bond vulnerable to attack by water.
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Figure 7

Distinct activities of Flp(R191A) and Flp(R308A) on an MeP-half-site. (A) Flp(R191A) cleaves the MeP-half-site (S) using Tyr-343 to form the protein–DNA adduct (revealed by SDS-PAGE; top) ( ). This intermediate is converted to the hydrolysis product (HP) (revealed by denaturing PAGE; bottom) in a subsequent slow reaction. Flp(R308A), by contrast, yields the hydrolysis product directly, without going through the MeP-tyrosyl intermediate ( ). (B) The binding of an Flp monomer to activates the scissile phosphate, leaving it exposed until the binding of a second Flp monomer delivers Tyr-343 to the active site in . (C) Concomitant with the binding of a Cre monomer to the LoxP site, Tyr-324 engages the scissile phosphate in , thus protecting it against direct water attack. (D) As vaccinia topoisomerase, like Cre, assembles its active site in , the scissile phosphate is protected at the strand cleavage step during DNA relaxation. However, the protein’s grip on DNA is loosened during the strand rotation step, leaving the phosphotyrosyl bond vulnerable to attack by water.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 8
Stepwise analysis of recombination by TPM. The DNA molecule containing two recombination target sites (open boxes) in head-to-head or head-to-tail orientation is attached to a glass slide at one end and tethered to a polystyrene bead at the other. The change in DNA length occurring at individual steps of recombination is reported by the corresponding changes in the BM amplitude of the bead (schematically indicated by the dashed lines with arrowheads at either end). The bending of the sites bound by the recombinase (shown as globules) causes a shortening of DNA, which is magnified upon synapsis. Chemical steps of recombination within the synapse can result in Holliday junction formation or completion of recombination (DNA excision in the case of head-to-tail sites and DNA inversion in the case of head-to-head sites). Upon dissociation of the recombinase from DNA by SDS treatment, the Holliday junction intermediate and the linear excision product will retain their low BM amplitude. The inversion product has the same length, and hence the same BM amplitude, as the starting DNA molecule.
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Figure 8

Stepwise analysis of recombination by TPM. The DNA molecule containing two recombination target sites (open boxes) in head-to-head or head-to-tail orientation is attached to a glass slide at one end and tethered to a polystyrene bead at the other. The change in DNA length occurring at individual steps of recombination is reported by the corresponding changes in the BM amplitude of the bead (schematically indicated by the dashed lines with arrowheads at either end). The bending of the sites bound by the recombinase (shown as globules) causes a shortening of DNA, which is magnified upon synapsis. Chemical steps of recombination within the synapse can result in Holliday junction formation or completion of recombination (DNA excision in the case of head-to-tail sites and DNA inversion in the case of head-to-head sites). Upon dissociation of the recombinase from DNA by SDS treatment, the Holliday junction intermediate and the linear excision product will retain their low BM amplitude. The inversion product has the same length, and hence the same BM amplitude, as the starting DNA molecule.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 9
Effect of synapse geometry on the BM amplitude of DNA. (A) The DNA contours for a pair of synapsed head-to-head sites are outlined for their alignment in parallel (left) or antiparallel (right) geometry. (B) Similar diagrams as in (A) represent the antiparallel (left) and parallel (right) synaptic configurations for head-to-tail sites. The effective length of DNA is slightly larger when its entry and exit points are at opposite ends of the synapse than when they are at the same end. For two DNA substrates that differ only in the relative orientation of the recombination sites, a difference in the BM amplitudes of synapsed head-to-head versus head-to-tail sites signifies a preferred geometry of the synapse.
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Figure 9

Effect of synapse geometry on the BM amplitude of DNA. (A) The DNA contours for a pair of synapsed head-to-head sites are outlined for their alignment in parallel (left) or antiparallel (right) geometry. (B) Similar diagrams as in (A) represent the antiparallel (left) and parallel (right) synaptic configurations for head-to-tail sites. The effective length of DNA is slightly larger when its entry and exit points are at opposite ends of the synapse than when they are at the same end. For two DNA substrates that differ only in the relative orientation of the recombination sites, a difference in the BM amplitudes of synapsed head-to-head versus head-to-tail sites signifies a preferred geometry of the synapse.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 10
Preferred antiparallel synapsis of a pair of tethered sites. (A) The two sites, whose orientation is indicated by the arrowheads, are constrained by a single-stranded tether (wavy line) to align only in the antiparallel geometry. The positions of the donor (Cy3) and acceptor (Cy5) fluorophores are indicated by the green and red circles, respectively. The shift towards lower FRET upon Flp(Y343F) binding is consistent with the synapsis of the sites as schematically diagrammed ( ). (B) In the tethered DNA substrate, analogous to that diagrammed in (A), the sites are constrained to pair only in the parallel geometry. Flp(Y343F) binding produces no change in FRET, suggesting the absence of parallel synapsis.
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Figure 10

Preferred antiparallel synapsis of a pair of tethered sites. (A) The two sites, whose orientation is indicated by the arrowheads, are constrained by a single-stranded tether (wavy line) to align only in the antiparallel geometry. The positions of the donor (Cy3) and acceptor (Cy5) fluorophores are indicated by the green and red circles, respectively. The shift towards lower FRET upon Flp(Y343F) binding is consistent with the synapsis of the sites as schematically diagrammed ( ). (B) In the tethered DNA substrate, analogous to that diagrammed in (A), the sites are constrained to pair only in the parallel geometry. Flp(Y343F) binding produces no change in FRET, suggesting the absence of parallel synapsis.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 11
The preferred assembly of one of two possible types of antiparallel synapse can specify the order in which strands are cleaved and exchanged during recombination. (A) A LoxP site bound by Cre is bent asymmetrically, the bend center being located close to one end of the strand exchange region. The two possible asymmetric bends would specify the cleavage of the bottom (blue) or the top strand (red). The scissile phosphates primed for cleavage are indicated by the filled circles; the quiescent ones are shown as open circles. For convenience of orienting the sites, the DNA arms are labeled as L (left) and R (right) as in Fig. 1 . (B) Based on the structure of the Cre-LoxP complex, fluorophores can be so positioned as to minimize donor (green)–acceptor (red) distance, and induce efficient FRET when the synapse favoring bottom strand cleavage (shown at the left) is assembled by Cre. In this fluorophore configuration, the FRET efficiency will be low for the synapse favoring top strand cleavage (shown at the right). (C) By reversing the left–right orientation of the fluorophores with respect to the strand exchange region, while maintaining their relative positioning, the synapsis favoring top strand cleavage (right) can be made to acquire the high FRET state. Experimental results indicate a clear preference for the synapse shown at the left in (B) suggesting that recombination is initiated by bottom strand cleavage and exchange ( ).
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Figure 11

The preferred assembly of one of two possible types of antiparallel synapse can specify the order in which strands are cleaved and exchanged during recombination. (A) A LoxP site bound by Cre is bent asymmetrically, the bend center being located close to one end of the strand exchange region. The two possible asymmetric bends would specify the cleavage of the bottom (blue) or the top strand (red). The scissile phosphates primed for cleavage are indicated by the filled circles; the quiescent ones are shown as open circles. For convenience of orienting the sites, the DNA arms are labeled as L (left) and R (right) as in Fig. 1 . (B) Based on the structure of the Cre-LoxP complex, fluorophores can be so positioned as to minimize donor (green)–acceptor (red) distance, and induce efficient FRET when the synapse favoring bottom strand cleavage (shown at the left) is assembled by Cre. In this fluorophore configuration, the FRET efficiency will be low for the synapse favoring top strand cleavage (shown at the right). (C) By reversing the left–right orientation of the fluorophores with respect to the strand exchange region, while maintaining their relative positioning, the synapsis favoring top strand cleavage (right) can be made to acquire the high FRET state. Experimental results indicate a clear preference for the synapse shown at the left in (B) suggesting that recombination is initiated by bottom strand cleavage and exchange ( ).

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 12
The magnitude of the DNA bend at the recombination target sites influences their localization within the branches of plectonemically supercoiled DNA. (A) The large DNA bend induced by Flp tends to localize presynaptic sites in separate plectonemic branches. Recombination between such sites yields topologically complex products. In the example shown, the excision reaction yields a 4-noded catenane. (B) The relatively small DNA bend induced by Cre tends to place presynaptic LoxP sites within the same plectonemic branch, thus simplifying the topology of recombination products. The excision reaction shown here yields two unlinked deletion circles.
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Figure 12

The magnitude of the DNA bend at the recombination target sites influences their localization within the branches of plectonemically supercoiled DNA. (A) The large DNA bend induced by Flp tends to localize presynaptic sites in separate plectonemic branches. Recombination between such sites yields topologically complex products. In the example shown, the excision reaction yields a 4-noded catenane. (B) The relatively small DNA bend induced by Cre tends to place presynaptic LoxP sites within the same plectonemic branch, thus simplifying the topology of recombination products. The excision reaction shown here yields two unlinked deletion circles.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 13
Unlinking of replication catenanes by XerCD-FtsK. (A) The unlinking of replication catenanes in is normally carried out by the type II topoisomerase Topo IV. For a 4-noded replication catenane containing parallel sites, unlinking by Topo IV will be completed in two steps (the straight path), removing two crossings at each step. Unlinking of the same catenane by FtsK-XerCD-mediated recombination at the sites requires four steps (the zigzag path), by removal of one crossing at a time. (B) The mechanisms for topology simplification by Topo IV and FtsK-XerCD are illustrated.
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Figure 13

Unlinking of replication catenanes by XerCD-FtsK. (A) The unlinking of replication catenanes in is normally carried out by the type II topoisomerase Topo IV. For a 4-noded replication catenane containing parallel sites, unlinking by Topo IV will be completed in two steps (the straight path), removing two crossings at each step. Unlinking of the same catenane by FtsK-XerCD-mediated recombination at the sites requires four steps (the zigzag path), by removal of one crossing at a time. (B) The mechanisms for topology simplification by Topo IV and FtsK-XerCD are illustrated.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 14
Tangle diagrams of attL- attR recombination performed by λ Int; ΔLk associated with DNA inversion. The λ Int mediated inversion reaction between attL and attR sites in three relaxed circular substrate molecules is represented by tangle diagrams (A-C). The O tangle contains inter-domainal DNA crossings trapped by Int (likely assisted by the accessory factors). O contains randomly trapped crossings in the ‘free’ DNA. The core recombination sites reside in the P tangle in anti-parallel geometry. The R tangle represents the post-recombination state of the sites. The tangle notations are shown at the top in bold; the corresponding DNA crossing (node) signs are given at the bottom in parentheses. The convention for the crossing signs (+1 or −1) is illustrated at the right, with the arrow heads denoting the direction (arbitrarily assigned) for the circular DNA axis. The simplest tangles (, , , ) are diagrammed at the far right. . In the DNA molecule shown here, one right-handed crossing is trapped in O, and none are contained in O. In tangle parlance, recombination changes P() tangle to the R() tangle, yielding an unknotted inversion circle. Note that a right-handed crossing in O in the substrate is by the tangle convention, but −1 by the sign convention. In the recombinant product, the crossing sign in O becomes +1 because of DNA inversion. The linking number change (ΔLk) accompanying the attL-attR reaction is +2. . The same reaction as in is shown for a molecule with two left-handed crossings present in O. The ΔLk for the reaction is −2. . A molecule performing the same reaction as in and is represented with P() and R() switched to P() and R(), respectively. The ΔLk associated with recombination is −2 in this case as well. The ΔLk changes are explained in more detail in the text.
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Figure 14

Tangle diagrams of attL- attR recombination performed by λ Int; ΔLk associated with DNA inversion. The λ Int mediated inversion reaction between attL and attR sites in three relaxed circular substrate molecules is represented by tangle diagrams (A-C). The O tangle contains inter-domainal DNA crossings trapped by Int (likely assisted by the accessory factors). O contains randomly trapped crossings in the ‘free’ DNA. The core recombination sites reside in the P tangle in anti-parallel geometry. The R tangle represents the post-recombination state of the sites. The tangle notations are shown at the top in bold; the corresponding DNA crossing (node) signs are given at the bottom in parentheses. The convention for the crossing signs (+1 or −1) is illustrated at the right, with the arrow heads denoting the direction (arbitrarily assigned) for the circular DNA axis. The simplest tangles (, , , ) are diagrammed at the far right. . In the DNA molecule shown here, one right-handed crossing is trapped in O, and none are contained in O. In tangle parlance, recombination changes P() tangle to the R() tangle, yielding an unknotted inversion circle. Note that a right-handed crossing in O in the substrate is by the tangle convention, but −1 by the sign convention. In the recombinant product, the crossing sign in O becomes +1 because of DNA inversion. The linking number change (ΔLk) accompanying the attL-attR reaction is +2. . The same reaction as in is shown for a molecule with two left-handed crossings present in O. The ΔLk for the reaction is −2. . A molecule performing the same reaction as in and is represented with P() and R() switched to P() and R(), respectively. The ΔLk associated with recombination is −2 in this case as well. The ΔLk changes are explained in more detail in the text.

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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Figure 15
Recombination-mediated cassette exchange. (A) In the classical RMCE, the replacement of a native locus by a donor DNA fragment is mediated by the same recombinase acting on two pairs of target sites ( and ) that are compatible only in one configuration of the DNA partners. The reaction may be followed by the replacement of one fluorescent reporter (RFP; red fluorescent protein) by another (GFP; green fluorescent protein). (B) In dual RMCE, the same reaction as in (A) is mediated by two separate recombinases acting on their respective cognate sites ( and ).
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Image of Figure 15
Figure 15

Recombination-mediated cassette exchange. (A) In the classical RMCE, the replacement of a native locus by a donor DNA fragment is mediated by the same recombinase acting on two pairs of target sites ( and ) that are compatible only in one configuration of the DNA partners. The reaction may be followed by the replacement of one fluorescent reporter (RFP; red fluorescent protein) by another (GFP; green fluorescent protein). (B) In dual RMCE, the same reaction as in (A) is mediated by two separate recombinases acting on their respective cognate sites ( and ).

Citation: Jayaram M, Ma C, Kachroo A, Rowley P, Guga P, Fan H, Voziyanov Y. 2015. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective, p 43-71. In Craig N, Chandler M, Gellert M, Lambowitz A, Rice P, Sandmeyer S (ed), Mobile DNA III. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.MDNA3-0021-2014
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