Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

Swan, M.K.; Bastia, Deepak; Davies, Christopher

doi:10.1073/pnas.0609046103

Cited by 31 publications

(42 citation statements)

References 46 publications

(74 reference statements)

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“…Recently, however, Swan et al published the crystal structure of the protein in complex with iteron DNA (19). Consistent with our results, their data also demonstrate the importance of amino acids Arg225 and Ser71 in DNA contact and replication and support DNA contact by Tyr74.…”

Section: Fig 3 Replication Inhibitor Function Of Variants (In Vivo)supporting

confidence: 81%

Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the π/DNA Interface

2007

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In previous work, we characterized the bases in an iteron of plasmid R6K that are important for the binding of protein monomers and dimers. Here we investigate the following six amino acids of , encoded by pir, hypothesized to be important for DNA contact: Ser71, Try74, Gly131, Gly211, Arg225, and Arg254.Rep proteins activate replication origins (ori's) by binding to tandem repeats in the DNA called iterons (3,8,13). When the sequences of iterons and rep genes are compared across families, striking similarities are observed (2,10,(16)(17)(18). A breakthrough in Rep/iteron studies came when Komori et al. published the crystal structure of a RepE monomer (from the F plasmid) in complex with its iteron DNA (10). The structure revealed that the Rep monomer contains two DNA-binding domains, an N-terminal WH1 (winged helix 1) and a C-terminal WH2 domain, with both domains used for iteron binding. A sequence alignment of several Reps presented by our group (10, 13) is a useful reference for identifying possible conserved amino acids in replication initiators, including RepE and the protein (described below). Based on the homologies observed in this class of proteins, Sharma et al. subsequently reported theoretical three-dimensional models for several Reps (18). The accuracy of these models remained to be tested.In a minimal R6K replicon, an ori called ␥ is activated by the Rep protein, , which is encoded by the pir gene ( Fig. 1A) (4,6,7,9,15). exists in both monomeric and dimeric forms, with monomers being activators of replication and dimers functioning both as replication inhibitors and as autorepressors of pir. The latter function is mediated by dimers binding to inverted repeats in the promoter that share sequence similarity with the iterons. monomers show little to no affinity for the inverted repeats but bind to an iteron better than dimers do (20). Insights into how achieves these similar but functionally distinct interactions were sought from analyses of Rep structure and the contacts that occur at the protein/DNA interface.In previous work from this lab, we determined the importance of individual bases in the iteron for the binding of monomers and dimers (14). Beyond this, we knew of no other experimental data addressing the contact residues of protein and DNA; a solved /iteron cocrystal structure was not available at that time. In the present study, we investigated the protein/DNA interface again, this time with an interest in identifying amino acids of that contact DNA. By analyzing the theoretical structural model of the monomer/iteron complex, we identified approximately 2 dozen amino acids as candidates for DNA contact and narrowed the list down to six for further study (Fig. 1B). Four (Ser71, Gly131, Arg225, and Arg254) appeared to be positionally conserved within the RepE/iteron cocrystal structure (Ser75, Gly125, Arg206, and Arg233, respectively). Tyr74 was strongly suspected to contact a thymine residue in the half of the iteron bound only by monomers (14). Lastly, Gly211 was of interest because it f...

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Section: Fig 3 Replication Inhibitor Function Of Variants (In Vivo)supporting

confidence: 81%

Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the π/DNA Interface

2007

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“…It is notable that the multiresistance RepA proteins contain a winged HTH, because structures of the F, R6K, and pPS10 RepA proteins encoded on plasmids harbored in Gram-negative bacteria also contain this motif (26,27). The winged HTH elements, however, are the only shared structural feature of these two Rep families; in contrast to pSK41 and pTZ2162 RepA, the F, R6K, and pPS10 RepA proteins consist of two tandem winged HTH domains and function as monomers (26,27).…”

Section: Resultsmentioning

confidence: 99%

“…The winged HTH elements, however, are the only shared structural feature of these two Rep families; in contrast to pSK41 and pTZ2162 RepA, the F, R6K, and pPS10 RepA proteins consist of two tandem winged HTH domains and function as monomers (26,27). Thus, these RepA proteins belong to distinct structural families.…”

Section: Resultsmentioning

confidence: 99%

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Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

Schumacher

Tonthat

Kwong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.RepA_N | replication initiation | S. aureus | two hybrid

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“…9,15-17 A π monomer contacts both the 5' half and the 3' half of the iteron through the C-terminal winged-helix (WH2) and the N-terminal winged helix (WH1), respectively, but a π dimer only contacts the 5' half of each iteron, including a highly conserved TGAGnG motif, with the WH2 of one of its subunits. [24][25][26][27] This potentially allows the WH2 motif of the second subunit of the dimer to contact another iteron-bearing sequence, 9,26 for instance, either of the other two functional oris of R6K, the α iteron or the β half-iteron, which are the active oris in vivo. 28,29 Such 'looping' is believed to transmit the replication signal from the internal iteron cluster at γ ori to the distant oris.…”

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confidence: 99%

Cooperative Binding Mode of the Inhibitors of R6K Replication, π Dimers

Bowers

Filutowicz

2008

Journal of Molecular Biology

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The replication initiator protein, π, plays an essential role in the initiation of replication of plasmid R6K. Both monomers and dimers of π bind to iterons in the γ origin of plasmid R6K, yet monomers facilitate open complex formation while dimers, the predominant form in the cell, do not. Consequently, π monomers activate replication while π dimers inhibit replication. Recently, it was shown that the monomeric form of π binds multiple tandem iterons in a strongly cooperative fashion, which might explain how monomers out-compete dimers for replication initiation when plasmid copy number and π supply are low. Here, we examine cooperative binding of π dimers and explore the role these interactions may have in the inactivation of γ origin. To examine π dimer/iteron interactions in the absence of competing π monomer/iteron interactions using wild-type π, constructs were made with key base changes to each iteron that eliminate π monomer binding yet have no impact on π dimer binding. Our results indicate that in the absence of π monomers, π dimers bind with greater cooperativity to alternate iterons than adjacent iterons, thus preferentially leaving intervening iterons unbound and the origin unsaturated. We discuss new insights into plasmid replication control by π dimers.It is believed that all naturally occurring plasmids employ efficient copy-control mechanisms to ensure their maintenance at a reasonably constant copy number from cell to cell. The antibiotic resistance plasmid, R6K, is maintained at a steady 15-20 copies per chromosome 1 in a wide variety of bacterial hosts. 2 For this to occur, regulatory controls at the step of replication initiation work to increase low plasmid copy numbers and reduce elevated ones. 3Controlled replication of plasmid R6K requires two plasmid-encoded elements: the iterons in the γ origin of replication (γ ori) and the pir gene that encodes the replication (Rep) protein, π ( Figure 1) 4-8 γ ori activation requires the binding of monomers of π protein to the seven 22 base pair (bp) iterons within γ ori that are adjacent to an A+T-rich region of the plasmid. 9-11 The binding of π monomers to iterons causes an apparent bending of the origin DNA, allowing the nearby A+T-rich region to melt, the replication complex to bind, and replication to start uni-directionally from a specific site within the A+T-rich region. 11-13While monomers of π activate replication, dimers of π appear to inhibit replication through several different mechanisms (Figure 1). 9,14-17 π dimers bind a non-iteron site within the A +T-rich region in proximity to the start sites for leading strand synthesis. 18 It has been hypothesized that π dimers negatively modulate the priming step of the replication process by *Corresponding author (M. Filutowicz):Tel. 608-262-6947; Fax. 608-262-9865; E-mail: msfiluto@facstaff.wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manu...

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Crystal structure of π initiator protein–iteron complex of plasmid R6K: Implications for initiation of plasmid DNA replication

Cited by 31 publications

References 46 publications

Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the π/DNA Interface

Structure-Based Functional Analysis of the Replication Protein of Plasmid R6K: Key Amino Acids at the π/DNA Interface

Mechanism of staphylococcal multiresistance plasmid replication origin assembly by the RepA protein

Cooperative Binding Mode of the Inhibitors of R6K Replication, π Dimers

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