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Chapter 19 : The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells

Authors: Yen-Ting Liu1, Saumitra Sau2, Chien-Hui Ma3, Aashiq H. Kachroo4, Paul A. Rowley5, Keng-Ming Chang6, Hsiu-Fang Fan7, Makkuni Jayaram8
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Affiliations: 1: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; 2: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; 3: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; 4: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; 5: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; 6: Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712; 7: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan; 8: Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan
Content Type: Monograph
Publication Year: 2015

Category: Clinical Microbiology

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Abstract:

Selfish genetic elements ( ), widespread in nature, are characterized by their ability to replicate efficiently and maintain themselves stably in host cell populations. A subset of these elements harbors the capacity to spread within a genome or, via horizontal transmission, between genomes. Selfish elements can also be frequently acquired by sexual transmission. The degree of selfishness can vary significantly among different elements. Some may increase the host’s fitness at least under certain conditions and, in doing so, add to their own fitness in a self-serving fashion. Others may be more decidedly selfish in that they contribute little toward the host’s fitness. Their long-term persistence is sustained solely by their capacity for replication and transmission during growth and division of host cells.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells
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Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
/content/10.1128/9781555818982.chap19.fig19-1
Figure 1
Genetic organization of the yeast plasmid and its copy number regulation. The double-stranded DNA genome of the yeast 2-micron plasmid is generally represented as a dumbbell-shaped molecule to highlight the 599-bp inverted repeat (the handle of the dumbbell) that separates two unique regions. The four coding regions harbored by the plasmid are , , , and . The directions in which these loci are transcribed are indicated by the arrowheads. The plasmid replication origin is indicated as . Flp is a site-specific recombinase, whose target sites (s) are embedded within the inverted repeat region. The plasmid partitioning locus can be divided into origin-proximal (-proximal) and origin-distal (-distal) segments. There are five repetitions of a 60-bp consensus sequence in -proximal. -distal, which harbors the termination signal for two origin-directed plasmid transcripts (1,650 and 700 nucleotides long) as well as a silencing element (shaded box), maintains -proximal as a transcription-free zone. The Rep1-Rep2- system ensures equal plasmid segregation. The (Flp-)-Raf1 system is responsible for the maintenance of the steady state plasmid copy number. The mechanism proposed for copy number correction of the plasmid by amplification ( ) invokes a Flp-mediated recombination event that changes the direction of one of the replication forks (indicated by the thin short arrows) with respect to the other during bidirectional replication of the plasmid. The ensuing dual unidirectional mode of replication amplifies the plasmid as a concatemer of tandem plasmid units. There is a marked asymmetry in the location of the sites (thick arrows) with respect to the replication origin (). The consequent difference in their replication status, one duplicated and the other not, is responsible for the relative inversion of the replication forks as a result of recombination between them. Efficient amplification without the danger of an unregulated increase in plasmid copy number is prevented by a transcriptional regulatory network. The putative [Rep1-Rep2] repressor negatively regulates , , and expression. Raf1 is thought to antagonize the action of the [Rep1-Rep2] repressor.
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Figure 1

Genetic organization of the yeast plasmid and its copy number regulation. The double-stranded DNA genome of the yeast 2-micron plasmid is generally represented as a dumbbell-shaped molecule to highlight the 599-bp inverted repeat (the handle of the dumbbell) that separates two unique regions. The four coding regions harbored by the plasmid are , , , and . The directions in which these loci are transcribed are indicated by the arrowheads. The plasmid replication origin is indicated as . Flp is a site-specific recombinase, whose target sites (s) are embedded within the inverted repeat region. The plasmid partitioning locus can be divided into origin-proximal (-proximal) and origin-distal (-distal) segments. There are five repetitions of a 60-bp consensus sequence in -proximal. -distal, which harbors the termination signal for two origin-directed plasmid transcripts (1,650 and 700 nucleotides long) as well as a silencing element (shaded box), maintains -proximal as a transcription-free zone. The Rep1-Rep2- system ensures equal plasmid segregation. The (Flp-)-Raf1 system is responsible for the maintenance of the steady state plasmid copy number. The mechanism proposed for copy number correction of the plasmid by amplification ( ) invokes a Flp-mediated recombination event that changes the direction of one of the replication forks (indicated by the thin short arrows) with respect to the other during bidirectional replication of the plasmid. The ensuing dual unidirectional mode of replication amplifies the plasmid as a concatemer of tandem plasmid units. There is a marked asymmetry in the location of the sites (thick arrows) with respect to the replication origin (). The consequent difference in their replication status, one duplicated and the other not, is responsible for the relative inversion of the replication forks as a result of recombination between them. Efficient amplification without the danger of an unregulated increase in plasmid copy number is prevented by a transcriptional regulatory network. The putative [Rep1-Rep2] repressor negatively regulates , , and expression. Raf1 is thought to antagonize the action of the [Rep1-Rep2] repressor.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 2
Aberrant plasmid amplification as a result of misregulation of Flp. Strand nicks formed at by the action of Flp, if unrepaired, will give rise to double-strand breaks when they encounter replication forks. Such a broken end can invade an intact circular plasmid and trigger aberrant plasmid amplification by repair synthesis. Posttranslational modification of Flp by sumoylation is important in preventing this mode of plasmid amplification ( ).
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Figure 2

Aberrant plasmid amplification as a result of misregulation of Flp. Strand nicks formed at by the action of Flp, if unrepaired, will give rise to double-strand breaks when they encounter replication forks. Such a broken end can invade an intact circular plasmid and trigger aberrant plasmid amplification by repair synthesis. Posttranslational modification of Flp by sumoylation is important in preventing this mode of plasmid amplification ( ).

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 3
Segregation of the 2-micron plasmid in the mutant. When the Ipl1 (aurora kinase) function is inactivated by a temperature-sensitive mutation, chromosomes frequently missegregate (shown here by the unequal DAPI staining in the mother and daughter compartments). A multicopy 2-micron reporter plasmid, fluorescence tagged by (GFP-LacI)-LacO interaction, also shows high missegregation under this condition ( ). Furthermore, the plasmid tends to be retained most often in the cell compartment that contains the bulk of the chromosomes. The segregation of a plasmid lacking (an plasmid) is not coupled to that of the chromosomes. However, such a plasmid missegregates frequently regardless of the Ipl1 status.
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Figure 3

Segregation of the 2-micron plasmid in the mutant. When the Ipl1 (aurora kinase) function is inactivated by a temperature-sensitive mutation, chromosomes frequently missegregate (shown here by the unequal DAPI staining in the mother and daughter compartments). A multicopy 2-micron reporter plasmid, fluorescence tagged by (GFP-LacI)-LacO interaction, also shows high missegregation under this condition ( ). Furthermore, the plasmid tends to be retained most often in the cell compartment that contains the bulk of the chromosomes. The segregation of a plasmid lacking (an plasmid) is not coupled to that of the chromosomes. However, such a plasmid missegregates frequently regardless of the Ipl1 status.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 4
Single copy reporter plasmids for segregation assays. Two types of single copy reporter plasmids have been developed. The copy number of a plasmid is maintained as one or nearly one by incorporating a into it. The can be conditionally inactivated by driving transcription through it from the inducible promoter. The reporter plasmid bordered by the target sites (shown by the arrowheads) for the R site-specific recombinase is integrated into a chromosome to keep the copy number strictly as one. The plasmid is excised by the inducible expression of the recombinase. Following inactivation of the plasmid-borne or recombination-mediated plasmid excision in G1 arrested cells, they are released into the cell cycle and plasmid segregation assayed at the anaphase stage. In either experimental scheme, the plasmid is visualized by operator-fluorescent repressor interaction.
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Figure 4

Single copy reporter plasmids for segregation assays. Two types of single copy reporter plasmids have been developed. The copy number of a plasmid is maintained as one or nearly one by incorporating a into it. The can be conditionally inactivated by driving transcription through it from the inducible promoter. The reporter plasmid bordered by the target sites (shown by the arrowheads) for the R site-specific recombinase is integrated into a chromosome to keep the copy number strictly as one. The plasmid is excised by the inducible expression of the recombinase. Following inactivation of the plasmid-borne or recombination-mediated plasmid excision in G1 arrested cells, they are released into the cell cycle and plasmid segregation assayed at the anaphase stage. In either experimental scheme, the plasmid is visualized by operator-fluorescent repressor interaction.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 5
Segregation of two single copy reporter plasmids. Two single copy reporter plasmids cohabiting a nucleus are distinguished by tagging one with green fluorescence [(GFP-LacI)-LacO] and the other with red fluorescence [(TetR-RFP)-TetO]. Under a functional Rep- system, the red and green plasmid sisters segregate most of the time in a one-to-one fashion to yield individual cells containing one red plasmid and one green plasmid ( ).
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Figure 5

Segregation of two single copy reporter plasmids. Two single copy reporter plasmids cohabiting a nucleus are distinguished by tagging one with green fluorescence [(GFP-LacI)-LacO] and the other with red fluorescence [(TetR-RFP)-TetO]. Under a functional Rep- system, the red and green plasmid sisters segregate most of the time in a one-to-one fashion to yield individual cells containing one red plasmid and one green plasmid ( ).

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 6
Segregation of a single copy reporter plasmid during monopolin-directed mitosis. The segregation of a fluorescence-tagged chromosome or a single copy reporter plasmid (p) is scored as 1:1 (equal segregation), 2:0 (mother-biased cosegregation), or 0:2 (daughter-biased cosegregation) during a normal or a monopolin-directed mitotic cell cycle. A subset of the missegregated plasmid sisters is often seen as coalesced or overlapping foci. The normal 1:1 segregation of sister chromatids is perturbed by monopolin toward 2:0 or 0:2 segregation. A similar effect is seen for p as well. In these radar plots, the degree of correlation between a reporter plasmid and a chromosome in their segregation patterns under the influence of monopolin is represented in terms of three variables: deviation from equal segregation (V′), tendency toward mother segregation (V′), and tendency toward daughter segregation (V′). The strong correlation between an plasmid and a chromosome in their bias-free cosegregation under the influence of monopolin is conveyed by the near congruence of the blue and green triangles. The absence of such correlation between an plasmid (lacking ) and a chromosome is evinced by the nonoverlapping disposition of the red triangle. Under conditions that uncouple the segregation of the 2-micron plasmid from that of the chromosomes, the tight correlation between an reporter plasmid and a reporter chromosome breaks down during monopolin directed mitosis. In the example shown here, the G1 to G2/M phase of the cell cycle is contrived to proceed in the absence of a functional spindle (by treatment with nocodazole). The metaphase arrested cells are washed free of nocodazole to permit spindle assembly and continuation of the cell cycle. Further details can be found in reference .
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Figure 6

Segregation of a single copy reporter plasmid during monopolin-directed mitosis. The segregation of a fluorescence-tagged chromosome or a single copy reporter plasmid (p) is scored as 1:1 (equal segregation), 2:0 (mother-biased cosegregation), or 0:2 (daughter-biased cosegregation) during a normal or a monopolin-directed mitotic cell cycle. A subset of the missegregated plasmid sisters is often seen as coalesced or overlapping foci. The normal 1:1 segregation of sister chromatids is perturbed by monopolin toward 2:0 or 0:2 segregation. A similar effect is seen for p as well. In these radar plots, the degree of correlation between a reporter plasmid and a chromosome in their segregation patterns under the influence of monopolin is represented in terms of three variables: deviation from equal segregation (V′), tendency toward mother segregation (V′), and tendency toward daughter segregation (V′). The strong correlation between an plasmid and a chromosome in their bias-free cosegregation under the influence of monopolin is conveyed by the near congruence of the blue and green triangles. The absence of such correlation between an plasmid (lacking ) and a chromosome is evinced by the nonoverlapping disposition of the red triangle. Under conditions that uncouple the segregation of the 2-micron plasmid from that of the chromosomes, the tight correlation between an reporter plasmid and a reporter chromosome breaks down during monopolin directed mitosis. In the example shown here, the G1 to G2/M phase of the cell cycle is contrived to proceed in the absence of a functional spindle (by treatment with nocodazole). The metaphase arrested cells are washed free of nocodazole to permit spindle assembly and continuation of the cell cycle. Further details can be found in reference .

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 7
A possible alternative mechanism for 2-micron plasmid amplification. A plasmid dimer may be formed by Flp-mediated recombination between two monomers. Resolution of the dimer by Flp during the act of replication will give rise to two interconnected circles being replicated iteratively by unidirectional replication forks. Such structures, named pince-nez molecules, have been observed by electron microscopy ( ).
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Figure 7

A possible alternative mechanism for 2-micron plasmid amplification. A plasmid dimer may be formed by Flp-mediated recombination between two monomers. Resolution of the dimer by Flp during the act of replication will give rise to two interconnected circles being replicated iteratively by unidirectional replication forks. Such structures, named pince-nez molecules, have been observed by electron microscopy ( ).

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 8
Flp-mediated site-specific recombination. The recombination reaction is initiated by the synapsis of two sites, L1-R1 and L2-R2 (L = left; R = right), each bound by two monomers of Flp (1, 2; 1′, 2′) across from the strand exchange region. The antiparallel arrangement of sites (left to right at the top and right to left at the bottom) within the recombination synapse is consistent with most (but not all) published data. The first pair of strand cleavage-exchange reactions gives rise to a Holliday junction intermediate; the second pair of analogous reactions resolves the junction into recombinant products, L1-R2 and L2-R1. The active Flp monomers, those adjacent to the scissile phosphates that are targeted by the active site tyrosine nucleophiles during a cleavage-exchange step, are shown in green. The switch between the active and inactive (magenta) pairs of Flp monomers accompanies the isomerization of the Holliday junction intermediate. The organization of key active site residues within the Flp active site is shown ( ). The conserved catalytic pentad of the tyrosine family corresponds to Arg-191, Lys-223, His-305, Arg-308, and Trp-330 in Flp. The active site tyrosine (Tyr-343) is delivered by a second Flp monomer.
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Figure 8

Flp-mediated site-specific recombination. The recombination reaction is initiated by the synapsis of two sites, L1-R1 and L2-R2 (L = left; R = right), each bound by two monomers of Flp (1, 2; 1′, 2′) across from the strand exchange region. The antiparallel arrangement of sites (left to right at the top and right to left at the bottom) within the recombination synapse is consistent with most (but not all) published data. The first pair of strand cleavage-exchange reactions gives rise to a Holliday junction intermediate; the second pair of analogous reactions resolves the junction into recombinant products, L1-R2 and L2-R1. The active Flp monomers, those adjacent to the scissile phosphates that are targeted by the active site tyrosine nucleophiles during a cleavage-exchange step, are shown in green. The switch between the active and inactive (magenta) pairs of Flp monomers accompanies the isomerization of the Holliday junction intermediate. The organization of key active site residues within the Flp active site is shown ( ). The conserved catalytic pentad of the tyrosine family corresponds to Arg-191, Lys-223, His-305, Arg-308, and Trp-330 in Flp. The active site tyrosine (Tyr-343) is delivered by a second Flp monomer.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 9
Chemical substitutions at the scissile phosphate position and half-site substrates for probing recombination mechanisms. When the 5′ bridging oxygen atom of the phosphodiester bond is replaced by sulfur, the significantly lower pKa of the 5′-thiol (compared to the 5′-hydroxyl) makes it a stronger leaving group. The 5′ thiolate substitution, in conjunction with active site mutants, can shed light on the general acid and/or accessory residues involved in facilitating leaving group departure. When one of the nonbridging oxygen atoms of the phosphate group is replaced by a methyl group, the resulting methylphosphonate (MeP) has no negative charge in the ground state. Furthermore, the methyl substitution introduces chirality at the phosphate center. The MeP substitution is useful for the analysis of electrostatic and stereochemical features of the recombination reaction. Half-site substrates simplify the recombination reaction while retaining its intrinsic chemical features. A half-site contains a single Flp binding element and a single scissile phosphate (or MeP) on the cleavable strand followed by three (or two) nucleotides of the strand exchange region. The modified scissile phosphate in the MeP half-site is indicated by the dot placed over the “p.” The other (noncleavable) strand contains all eight nucleotides of the strand exchange region. When the 5′-hydroxyl group of this strand is phosphorylated, it is blocked from partaking in a strand joining reaction. Tyr-343 mediated cleavage of the MeP bond will give rise to the Flp-linked DNA intermediate, which can be hydrolyzed slowly over time. Cleavage within a half-site is nearly irreversible, as the short tri- or dinucleotide product diffuses away from the reaction center. Since Flp-bound half-sites can associate to form dimers, trimers, and tetramers, Tyr-343 can be donated in as the cleavage nucleophile. In principle, the same hydrolysis product can also be formed by direct attack of water on the MeP bond (shown at the left). However, such a reaction is not observed during the action of Flp on native DNA substrates containing an unmodified phosphate at the scissile position.
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Figure 9

Chemical substitutions at the scissile phosphate position and half-site substrates for probing recombination mechanisms. When the 5′ bridging oxygen atom of the phosphodiester bond is replaced by sulfur, the significantly lower pKa of the 5′-thiol (compared to the 5′-hydroxyl) makes it a stronger leaving group. The 5′ thiolate substitution, in conjunction with active site mutants, can shed light on the general acid and/or accessory residues involved in facilitating leaving group departure. When one of the nonbridging oxygen atoms of the phosphate group is replaced by a methyl group, the resulting methylphosphonate (MeP) has no negative charge in the ground state. Furthermore, the methyl substitution introduces chirality at the phosphate center. The MeP substitution is useful for the analysis of electrostatic and stereochemical features of the recombination reaction. Half-site substrates simplify the recombination reaction while retaining its intrinsic chemical features. A half-site contains a single Flp binding element and a single scissile phosphate (or MeP) on the cleavable strand followed by three (or two) nucleotides of the strand exchange region. The modified scissile phosphate in the MeP half-site is indicated by the dot placed over the “p.” The other (noncleavable) strand contains all eight nucleotides of the strand exchange region. When the 5′-hydroxyl group of this strand is phosphorylated, it is blocked from partaking in a strand joining reaction. Tyr-343 mediated cleavage of the MeP bond will give rise to the Flp-linked DNA intermediate, which can be hydrolyzed slowly over time. Cleavage within a half-site is nearly irreversible, as the short tri- or dinucleotide product diffuses away from the reaction center. Since Flp-bound half-sites can associate to form dimers, trimers, and tetramers, Tyr-343 can be donated in as the cleavage nucleophile. In principle, the same hydrolysis product can also be formed by direct attack of water on the MeP bond (shown at the left). However, such a reaction is not observed during the action of Flp on native DNA substrates containing an unmodified phosphate at the scissile position.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 10
Activities of Flp(R191A) and Flp(R308A) on an MeP substituted half-site substrate; the difference between - and - active sites in protecting the scissile phosphate from abortive hydrolysis. The MeP half-site reactions are analyzed by electrophoresis in SDS-polyacrylamide gels (top panels) to detect the Flp linked tyrosyl-DNA intermediate or in urea-polyacrylamide gels (bottom panels) to visualize the hydrolysis product (HP). The unreacted substrate band is indicated by “S.” The asterisk marks the P label placed at the 5′ end of the cleavable strand of the half-site. Flp(R191A) forms the tyrosyl intermediate, which then undergoes slow hydrolysis. Flp(R308A) does not form the tyrosyl intermediate. Instead, it promotes direct hydrolysis of the MeP bond. When a cis-acting recombinase (Cre, for example) monomer binds to its target site, the tyrosine nucleophile is oriented within the active site to engage the scissile phosphate. The binding of a second monomer allosterically activates this active site to trigger tyrosine-mediated strand cleavage. Thus, the scissile phosphate is not prone to attack by water acting as the nucleophile. Binding of a Flp monomer activates the adjacent scissile phosphate even though the tyrosine nucleophile is not oriented within the active site. The engagement of the scissile phosphate by tyrosine must await the binding of a second Flp monomer. The time lag between the two binding events renders the scissile phosphate susceptible to direct hydrolysis. Electrostatic repulsion of water by Arg-308 appears to prevent this abortive reaction.
10.1128/9781555818982/fig19-10_thmb.gif
10.1128/9781555818982/fig19-10.gif
Image of Figure 10
Figure 10

Activities of Flp(R191A) and Flp(R308A) on an MeP substituted half-site substrate; the difference between - and - active sites in protecting the scissile phosphate from abortive hydrolysis. The MeP half-site reactions are analyzed by electrophoresis in SDS-polyacrylamide gels (top panels) to detect the Flp linked tyrosyl-DNA intermediate or in urea-polyacrylamide gels (bottom panels) to visualize the hydrolysis product (HP). The unreacted substrate band is indicated by “S.” The asterisk marks the P label placed at the 5′ end of the cleavable strand of the half-site. Flp(R191A) forms the tyrosyl intermediate, which then undergoes slow hydrolysis. Flp(R308A) does not form the tyrosyl intermediate. Instead, it promotes direct hydrolysis of the MeP bond. When a cis-acting recombinase (Cre, for example) monomer binds to its target site, the tyrosine nucleophile is oriented within the active site to engage the scissile phosphate. The binding of a second monomer allosterically activates this active site to trigger tyrosine-mediated strand cleavage. Thus, the scissile phosphate is not prone to attack by water acting as the nucleophile. Binding of a Flp monomer activates the adjacent scissile phosphate even though the tyrosine nucleophile is not oriented within the active site. The engagement of the scissile phosphate by tyrosine must await the binding of a second Flp monomer. The time lag between the two binding events renders the scissile phosphate susceptible to direct hydrolysis. Electrostatic repulsion of water by Arg-308 appears to prevent this abortive reaction.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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Figure 11
Single molecule TPM analysis of Flp site-specific recombination. The substrate DNA molecule containing a pair of sites in direct (deletion substrate) or inverted (inversion substrate) orientation is tethered to a glass surface at one end and to a polystyrene bead at the other. The effective length of the DNA changes as a result of Flp binding to sites or synapsis of the bound sites. Such changes can be assayed by changes in the Brownian motion (BM) amplitude of the attached bead (indicated schematically by the dashed line with arrowheads at either end). Within the recombination synapse, the chemical steps of recombination may or may not occur. The fate of the synapsed molecules can be revealed by the BM amplitudes they display after being stripped of noncovalently associated Flp. The expected BM amplitudes following the addition of Flp to a deletion substrate. Binding of Flp to the sites (unfilled rectangular boxes on the tethered DNA molecule) will cause a slight reduction in the BM amplitude because of the DNA bending induced by Flp. A more marked reduction in BM amplitude follows upon synapsis of the sites. This low BM amplitude will be retained by the Holliday junction intermediate and the linear product of excision even after the addition of SDS. Flp-bound molecules that fail to synapse (nonproductive complexes) or synapsed molecules that fail to recombine (wayward complexes) will return to the high BM amplitude of the starting DNA substrate after SDS challenge. In the case of the inversion substrate, SDS challenge cannot distinguish a wayward complex from a completed recombination event. This is because the length of the inversion product is the same as that of the parental substrate. Time traces of individual molecules illustrating two different states of the deletion substrate following Flp addition are shown. The trace in the left panel indicates a molecule that formed a stable synapse of the Flp-bound sites. The low BM amplitude of this molecule after SDS challenge indicates that it underwent Holliday junction formation or a complete recombination event. The trace in the right panel indicates a molecule that underwent Flp binding and synapsis but failed to recombine or to form the Holliday junction, as indicated by its return to the starting high BM amplitude after SDS treatment (a wayward complex). The horizontal stippled bar represents the BM amplitude of the DNA in the synapsed state of sites.
10.1128/9781555818982/fig19-11_thmb.gif
10.1128/9781555818982/fig19-11.gif
Image of Figure 11
Figure 11

Single molecule TPM analysis of Flp site-specific recombination. The substrate DNA molecule containing a pair of sites in direct (deletion substrate) or inverted (inversion substrate) orientation is tethered to a glass surface at one end and to a polystyrene bead at the other. The effective length of the DNA changes as a result of Flp binding to sites or synapsis of the bound sites. Such changes can be assayed by changes in the Brownian motion (BM) amplitude of the attached bead (indicated schematically by the dashed line with arrowheads at either end). Within the recombination synapse, the chemical steps of recombination may or may not occur. The fate of the synapsed molecules can be revealed by the BM amplitudes they display after being stripped of noncovalently associated Flp. The expected BM amplitudes following the addition of Flp to a deletion substrate. Binding of Flp to the sites (unfilled rectangular boxes on the tethered DNA molecule) will cause a slight reduction in the BM amplitude because of the DNA bending induced by Flp. A more marked reduction in BM amplitude follows upon synapsis of the sites. This low BM amplitude will be retained by the Holliday junction intermediate and the linear product of excision even after the addition of SDS. Flp-bound molecules that fail to synapse (nonproductive complexes) or synapsed molecules that fail to recombine (wayward complexes) will return to the high BM amplitude of the starting DNA substrate after SDS challenge. In the case of the inversion substrate, SDS challenge cannot distinguish a wayward complex from a completed recombination event. This is because the length of the inversion product is the same as that of the parental substrate. Time traces of individual molecules illustrating two different states of the deletion substrate following Flp addition are shown. The trace in the left panel indicates a molecule that formed a stable synapse of the Flp-bound sites. The low BM amplitude of this molecule after SDS challenge indicates that it underwent Holliday junction formation or a complete recombination event. The trace in the right panel indicates a molecule that underwent Flp binding and synapsis but failed to recombine or to form the Holliday junction, as indicated by its return to the starting high BM amplitude after SDS treatment (a wayward complex). The horizontal stippled bar represents the BM amplitude of the DNA in the synapsed state of sites.

Citation: Liu Y, Sau S, Ma C, Kachroo A, Rowley P, Chang K, Fan H, Jayaram M. 2015. The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells, p 325-347. In Tolmasky M, Alonso J (ed), Plasmids: Biology and Impact in Biotechnology and Discovery. ASM Press, Washington, DC. doi: 10.1128/microbiolspec.PLAS-0003-2013
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