Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

Pai, Karnire S.; Bussiere, Dirksen E.; Wang, Fenggang; White, Stephen W.; Bastia, Deepak

doi:10.1073/pnas.93.20.10647

Cited by 20 publications

(23 citation statements)

References 26 publications

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“…We have also constructed a three-dimensional model of the RTP-bipolar Ter complex by combining the crystal structure of the apoprotein (5) and the affinity cleavage data. The model that resulted is consistent with mutagenesis and cross-linking data that suggested roles for the ␣3 helix, the ␤2 strand, and the N-terminal arm of RTP in DNA binding (10,21). Thus the ␣3 helix appears to be the recognition helix that invades the major groove, and the ␤2 strand makes minor groove contacts.…”

Section: Discussionsupporting

confidence: 79%

“…Instead, additional Cys residues were introduced one at a time into the locations indicated (see Figs. 3 and 4) by site-directed mutagenesis (QuickChange kit, Stratagene), and the residues were derivatized with EPD and cleavage reactions were performed as published (8,10). When DNA cleavage is catalyzed by an Fe-EDTA-conjugated DNA-binding protein, cleavage occurs at the C1Ј and/or C4Ј bonds of the sugar moieties within 3-4 Å from the location of the hydroxyradical generator, i.e.…”

Section: Conversion Of Rtp To a Chemical Nuclease And Cleavage Maps Omentioning

confidence: 99%

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Structural and Functional Analysis of a Bipolar Replication Terminus

Mohanty

Bussiere²,

Sahoo

et al. 2001

Journal of Biological Chemistry

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We have delineated the amino acid to nucleotide contacts made by two interacting dimers of the replication terminator protein (RTP) of Bacillus subtilis with a novel naturally occurring bipolar replication terminus by converting RTP to a site-directed chemical nuclease and mapping its cleavage sites on the terminus. The data show a relatively symmetrical arrangement of the amino acid to base contacts, and a comparison of the bipolar contacts with that of a normal unipolar terminus suggests that the DNA-protein contacts play an important determinative role in generating polarity from structurally symmetrical RTP dimers. The amino acid to nucleotide contacts provided distance constraints that enabled us to build a three-dimensional model of the protein-DNA complex. The model is consistent with features of the bipolar Ter⅐RTP complex derived from mutational and cross-linking data. The bipolar terminus arrested Escherichia coli DNA replication and DnaB helicase and T7 RNA polymerase in vitro in both orientations. RTP arrested the unwinding of duplex DNA on the bipolar Ter DNA substrate regardless of the length of the duplex DNA. The latter result suggested further that the terminus arrested authentic DNA unwinding by the helicase rather than just translocation of helicase on DNA.In many prokaryotic and some eukaryotic replicons, replication forks initiated at specific replication origins and moving bi-directionally are not terminated randomly but in regions delimited by polar replication termini (Ter).1 The Ter sites are usually short sequences that specifically bind to a replication terminator protein (RTP) and arrest fork moving in only one direction with respect to the origin but not the other. The Ter sites are usually located in two clusters of opposite polarity in such a way that the forks moving clockwise on a circular chromosome pass through the first cluster (that has the nonarresting polarity) and are arrested at the termini of the second cluster, which has blocking polarity. The same is true for forks moving in a counterclockwise direction (1, 2).Each Ter site of Bacillus subtilis binds to two interacting dimers of RTP to arrest forks in a polar mode (3, 4). Thus, there are two interesting mechanistic questions to be addressed in this context. First, how is the polarity of fork arrest generated, considering the fact that the dimeric RTP has a symmetrical structure (5). Second, what is the molecular mechanism of fork arrest (see reviews in Refs. 2 and 6). In this study we have endeavored to address the first question.We have approached the question by determining the amino acid to nucleotide contacts of RTP bound to the novel naturally occurring bipolar terminus of the plasmid pLS20 (7) and comparing and contrasting the results with similar contacts derived from a normal unipolar Ter site. We have converted RTP to a site-directed chemical nuclease by coupling it to an organic Fe-EDTA conjugate at certain rationally selected critical amino acid residues that are known to be involved in contacting Ter DNA (8...

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Section: Discussionsupporting

confidence: 79%

Section: Conversion Of Rtp To a Chemical Nuclease And Cleavage Maps Omentioning

confidence: 99%

Structural and Functional Analysis of a Bipolar Replication Terminus

Mohanty

Bussiere²,

Sahoo

et al. 2001

Journal of Biological Chemistry

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“…and is known to contact the major groove of Ter DNA (10,26). This derivative was therefore used as a positive control.…”

Section: Resultsmentioning

confidence: 99%

A Single Domain of the Replication Termination Protein ofBacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase

Gautam

Mulugu

Alexander

et al. 2001

Journal of Biological Chemistry

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The current models that have been proposed to explain the mechanism of replication termination are ( DNA replication in many prokaryotic chromosomes and at some eukaryotic chromosome regions is arrested at specific sequences called replication termini or Ter sites (6). Ter sites act as polar barriers to fork movement and act essentially as replication traps. In Bacillus subtilis, the Ter DNA interacts with a sequence-specific DNA-binding protein called replication terminator protein (RTP) 1 that acts as a polar contrahelicase; i.e. it impedes helicase-catalyzed DNA unwinding when present in one orientation, whereas it lets the helicase pass through unimpeded in the opposite orientation (1-4).The bacterial chromosome is believed to exist in vivo not as naked DNA but as a DNA-protein complex, with most of the DNA-binding proteins remaining bound to the chromosome (5). Despite the fact that some of these proteins bind to DNA with relatively high affinity (e.g. lac repressor), the replication fork apparently has the ability to pass through these complexes unimpeded. The only region of the chromosome that is known to arrest effectively the replication forks is the terminus (6). The preceding observations suggest the following: (i) the replication apparatus apparently has an activity that allows it to pass through most protein-DNA complexes, some of which contain strong DNA-binding proteins, and (ii) since the replication terminus is able to arrest forks effectively, the terminator protein-DNA complex is likely to have special features that enable it to arrest replication forks. Thus, high affinity binding of terminator protein to Ter sites per se does not appear to be sufficient to cause the replication-terminating activity of RTP (12). We have hypothesized that DnaB (or the equivalent helicase of B. subtilis)-RTP interaction plays a key role in fork arrest (1). Despite the existence of in vitro evidence for RTPDnaB interaction (7, 12), the validity of the protein-protein interaction model has been debated (11).The raison d'etre for carrying out this work was 2-fold: (i) to perform additional experiments, using independent approaches and different mutant forms of RTP, to reexamine the question of RTP-DnaB interaction in vitro and (ii) to investigate whether a common domain of RTP is involved in the arrest of both RNA polymerase and helicase. The observations presented here confirm the biologically meaningful interaction between RTP and DnaB and further extend the result by showing that a common domain of RTP seems to be involved in the arrest of both DnaB helicase and T7 RNA polymerase (and perhaps other RNA polymerases).The replication termini of B. subtilis ( Fig. 1) consist of overlapping core and auxiliary sites. A RTP dimer first binds to a core and then, by cooperativity, promotes the binding of a second dimer of RTP to the auxiliary site (8,9). Interaction between two dimers is essential for fork arrest with the core end of the Ter site arresting the helicase and the auxiliary end, allowing the helicase to p...

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“…This was followed quickly by models for the structures of the complex of the RTP dimer and tetramer with half and full Ter sites, derived from consolidation of the structure of the free protein with an extensive series of biochemical data (115,125,134,135). The structure of the half-site complex determined subsequently by a combination of nuclear magnetic resonance and crystallographic studies (172) was largely in accord with these models.…”

Section: The Crystal Structure Of the Tus-ter Complexmentioning

confidence: 99%

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

Neylon

Kralicek

Hill

et al. 2005

Microbiol Mol Biol Rev

134

144

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The arrest of DNA replication in Escherichia coli is triggered by the encounter of a replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is sufficient to block the helicase in a polar manner, without the need for specific protein-protein interactions. This review integrates three decades of experimental information on the action of the Tus-Ter complex with information available from the Tus-TerA crystal structure. We conclude that while it is possible to explain polar fork arrest by a mechanism involving only the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB interactions. The evidence suggests, therefore, that the termination system is more subtle and complex than may have been assumed. We describe some further experiments and insights that may assist in unraveling the details of this fascinating process

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Structure of the replication terminus-terminator protein complex as probed by affinity cleavage.

Cited by 20 publications

References 26 publications

Structural and Functional Analysis of a Bipolar Replication Terminus

Structural and Functional Analysis of a Bipolar Replication Terminus

A Single Domain of the Replication Termination Protein ofBacillus subtilis Is Involved in Arresting Both DnaB Helicase and RNA Polymerase

Replication Termination inEscherichia coli: Structure and Antihelicase Activity of the Tus-TerComplex

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