P1 Partition Complex Assembly Involves Several Modes of Protein-DNA Recognition

Vecchiarelli, Anthony G.; Schumacher, Maria A.; Funnell, Barbara E.

doi:10.1074/jbc.m611250200

Cited by 16 publications

(12 citation statements)

References 31 publications

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“…The structure also confirms that the primary binding of the protein involves the central helix-turn-helix motif of ParB contacting the BoxA residues of parS. The bifunctional binding and the flexibility of the ParB protein may allow the formation of a number of alternative complexes either by spanning the two arms of the site that are brought close together by IHF bending or by spanning the parS sites of two different plasmids in the pairing action thought to be important for partition (7,10,19,23).…”

supporting

confidence: 52%

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Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements

Dabrazhynetskaya

Brendler

et al. 2009

J Bacteriol

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The P1, P7, and pMT1 par systems are members of the P1 par family of plasmid partition elements. Each has a ParA ATPase and a ParB protein that recognizes the parS partition site of its own plasmid type to promote the active segregation of the plasmid DNA to daughter cells. ParB contacts two parS motifs known as BoxA and BoxB, the latter of which determines species specificity. We found that the substitution of a single orthologous amino acid in ParB for that of a different species has major effects on the specificity of recognition. A single change in ParB can cause a complete switch in recognition specificity to that of another species or can abolish specificity. Specificity changes do not necessarily correlate with changes in the gross DNA binding properties of the protein. Molecular modeling suggests that species specificity is determined by the capacity to form a hydrogen bond between ParB residue 288 and the second base in the BoxB sequence. As changes in just one ParB residue and one BoxB base can alter species specificity, plasmids may use such simple changes to evolve new species rapidly.The P1 plasmid partition system involves interactions between three proteins (P1 ParA and ParB and the host integration host factor [IHF]) and a ca. 80-bp DNA site (P1 parS), which results in the physical movement of the plasmid DNA molecules in a coordinated fashion (13). Similar elements are found in a variety of plasmid types from various bacterial species (9). Although very similar, the P1 par, pMT1 par, and P7 par members each show unique partition specificities: the Par proteins of one species fail to function with the parS sites of the other two. These different par species are compatible with each other; they do not promote the incompatibility seen between pairs of plasmids that carry identical par elements (4).The Par proteins of the P1 plasmid will not support P7 plasmid partition and vice versa. A key interaction in determining this species specificity proved to be an interaction between the ParB protein and the two BoxB repeat sequences that form the boundaries of the parS site. When the sequences of these P1 parS boxes were changed to their P7 equivalents, the site switched specificity and responded only to the P7 proteins (11). This observation was extended to show that the BoxB sequence also determined species specificity in the pMT1 parS site and showed that the specificity of a parS site could be changed to that of another species by as little as a single BoxB base change (4). Domain-swapping experiments showed that the critical protein sequence for specificity, a 16-amino-acid stretch termed the discriminator recognition sequence (DRS), is located near the carboxyl terminus of the ParB protein (19). This forms a secondary contact between ParB and the parS site, the primary contact involving a helix-turn-helix motif in the central region of ParB and the BoxA sequences in parS (22). The primary interactions are essentially the same in each of the plasmid species.

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confidence: 52%

“…The crystal structure of P1 ParB in contact with parS DNA has been solved (23). This structure shows that ParB residues Lys287 to Lys309 of the DRS are in contact with the BoxB bases by a previously unknown type of DNA binding interaction.…”

mentioning

confidence: 98%

Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements

Dabrazhynetskaya

Brendler

et al. 2009

J Bacteriol

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“…The P1par locus is an operon producing ParA and ParB proteins followed directly by the centromere analog site, parS (9). The ParB protein binds specifically to the parS site (35). The ParA protein is an ATPase that interacts with the ParB-parS complex (3,4,11).…”

mentioning

confidence: 99%

P1 Plasmid Segregation: Accurate Redistribution by Dynamic Plasmid Pairing and Separation

et al. 2010

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Low-copy-number plasmids, such as P1 and F, encode a type Ia partition system (P1par or Fsop) for active segregation of copies to daughter cells. Typical descriptions show a single central plasmid focus dividing and the products moving to the cell quarter regions, ensuring segregation. However, using improved optical and analytical tools and large cell populations, we show that P1 plasmid foci are very broadly distributed. Moreover, under most growth conditions, more than two foci are frequently present. Each focus contains either one or two plasmid copies. Replication and focus splitting occur at almost any position in the cell. The products then move rapidly apart for approximately 40% of the cell length. They then tend to maintain their relative positions. The segregating foci often pass close to or come to rest close to other foci in the cell. Foci frequently appear to fuse during these encounters. Such events occur several times in each cell and cell generation on average. We argue that foci pair with their neighbors and then actively separate again. The net result is an approximately even distribution of foci along the long cell axis on average. We show mathematically that trans-pairing and active separation could greatly increase the accuracy of segregation and would produce the distributions of foci that we observe. Plasmid pairing and separation may constitute a novel fine-tuning mechanism that takes the basic pattern created when plasmids separate after replication and converts it to a roughly even pattern that greatly improves the fidelity of plasmid segregation.

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“…Although the proteins and the cis elements differ between these two systems, the proteins that participate in the partition of these plasmids have similar functions and conserved sequences (2,39). In P1, the cis element that is required for plasmid partition, parS, contains two sets of repeat sequences, which are recognized by a partition protein, ParB (45). Between the repeats is an integration host factor-binding site (17).…”

mentioning

confidence: 99%

Stabilization of pSW100 fromPantoea stewartiiby the F Conjugation System

Lin

Liu

2008

J Bacteriol

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Plasmid pSW100 is 1 of the 13 plasmids from Pantoea stewartii subsp. stewartii SW2 which has a replicon that resembles that of ColE1. This work uses a pSW100 derivative, pSW140K, to study how the pSW100 replicon is stably maintained in its hosts. Our results indicate that although pSW140K is stable in Escherichia coli HB101, the plasmid is rapidly lost in another E. coli strain, DH5␣, indicating that the genetic background of an E. coli strain affects the stability of pSW140K. Mutagenesis of E. coli HB101 with EZ::TN revealed that mutations in traC, traF, traG, traN, and traV, which encode the components of the sex pilus assembly, reduce plasmid stability. Furthermore, this work identified that a 38-bp region located immediately upstream of the RNAII promoter is critical to the maintenance of plasmid stability in E. coli HB101. TraC binds to the region, and in addition, deleting the region destabilizes the plasmid. Furthermore, inserting this 38-bp fragment into a plasmid that contains the minimal replicon from pSW200 stabilizes the plasmid in E. coli HB101. Fluorescence in situ hybridization and immunofluorescence staining also revealed that derivatives of pSW100, pSW128A, and TraC are colocalized in cells, suggesting that pSW100 may use the sex pilus assembly as a partition apparatus to ensure the even distribution of the plasmid during cell division, which may thus maintain the plasmid's stability.

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P1 Partition Complex Assembly Involves Several Modes of Protein-DNA Recognition

Cited by 16 publications

References 31 publications

Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements

Switching Protein-DNA Recognition Specificity by Single-Amino-Acid Substitutions in the P1parFamily of Plasmid Partition Elements

P1 Plasmid Segregation: Accurate Redistribution by Dynamic Plasmid Pairing and Separation

Stabilization of pSW100 fromPantoea stewartiiby the F Conjugation System

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