DNA-protein interaction at the replication termini of plasmid R6K.

Sista, Prakash; Hutchinson, Catherine; Bastia, Deepak

doi:10.1101/gad.5.1.74

Cited by 34 publications

(36 citation statements)

References 32 publications

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“…Although both our in vitro results reported here and our in vivo data (32) showed that a C6 to A6 in transversion in Ter did not manifest any loss of arrest of a sliding or unwinding DnaB, another group (33) has reported that the same mutation can cause a 3-fold reduction in fork arrest. This incremental reduction in fork arrest perhaps can be reconciled with other observations as discussed below.…”

Section: Discussioncontrasting

confidence: 68%

Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase

Bastia

Zzaman²,

Krings³

et al. 2008

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

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The replication terminator protein Tus of Escherichia coli promotes polar fork arrest at sequence-specific replication termini (Ter) by antagonizing DNA unwinding by the replicative helicase DnaB. Here, we report that Tus is also a polar antitranslocase. We have used this activity as a tool to uncouple helicase arrest at a Tus-Ter complex from DNA unwinding and have shown that helicase arrest occurred without the generation of a DNA fork or a bubble of unpaired bases at the Tus-Ter complex. A mutant form of Tus, which reduces DnaB-Tus interaction but not the binding affinity of Tus for Ter DNA, was also defective in arresting a sliding DnaB. A model of polar fork arrest that proposes melting of the Tus-Ter complex and flipping of a conserved C residue of Ter at the blocking but not the nonblocking face has been reported. The model suggests that enhanced stability of Tus-Ter interaction caused by DNA melting and capture of a flipped base by Tus generates polarity strictly by enhanced protein-DNA interaction. In contrast, the observations presented here show that polarity of helicase and fork arrest in vitro is generated by a mechanism that not only involves interaction between the terminator protein and the arrested enzyme but also of Tus with Ter DNA, without any melting and base flipping in the termination complex.protein-DNA interaction ͉ protein-protein interaction ͉ site-directed interstrand cross-linking T he replication of DNA in many prokaryotes and in certain regions of eukaryotic chromosomes is specifically terminated at specialized sequences called replication termini (Ter) ( Fig. 1 A and B) that cause orientation-dependent fork arrest, and such arrest performs important physiological functions (1-3). In eukaryotes, sequence-specific replication termini are not present within every replication unit. Instead, the termini are found at specialized locations such as the nontranscribed spacers of rDNA (4) and at the mating type switch locus of Schizosaccharomyces pombe (5). We and others have shown by in vitro analyses that the replication termination proteins of Escherichia coli and Bacillus subtilis are polar contrahelicases, i.e., the proteins cause unidirectional arrest of the replicative helicase DnaB upon binding to the Ter sequences (6-10). The crystal structures of the replication terminator protein (RTP) of B. subtilis (11) and that of E. coli, called Tus (12), have been solved. Despite the fact that the proteins have very different structures, both proteins interact in vitro with their cognate binding sites to arrest DnaB helicase and RNA polymerase of E. coli in a polar mode (10, 13).A satisfactory model of polar fork arrest should take into account the following biological observations. First, a Tus-Ter complex arrests only some helicases such as DnaB but not others such as PcrA, helicase I, and UvrD helicase (9, 14) in vitro. In fact, in vivo genetic experiments show that UvrD helicase removes Tus protein from Ter sites (15). Further evidence of helicase specificity is indicated by the observat...

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Section: Discussioncontrasting

confidence: 68%

Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase

Bastia

Zzaman²,

Krings³

et al. 2008

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

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“…9) is now predicted for a complex that is in equilibrium between a specifically bound form and a nonspecifically bound form. Furthermore, this suggests that such sites could spread farther in cases where the specific binding is weaker, such as with TerR2, as is observed (54,158). This can also explain an apparent discrepancy between results on the effects of the R198A mutation.…”

Section: Prospects For the Futurementioning

confidence: 79%

“…3), both groups reported protected sites outside this region. Sista et al (158) found four such sites, between 1 and 3 base pairs preceding and 1 following the Tus-binding site in the TerR2 sequence (Fig. 9).…”

Section: Dna Modification and Protection Studiesmentioning

confidence: 94%

“…Ethylation interference showed that the phosphates between G10 and T14 (on the top strand as shown in Fig. 9) as well as those between A18 and C13 (on the bottom strand) were necessary for Tus binding (54,158). The phosphates of all these nucleotides interact with Tus in the crystal structure (Fig.…”

Section: Dna Modification and Protection Studiesmentioning

confidence: 99%

“…Tus was shown by footprinting with copper-phenanthroline (159), DNase I (65,130), and hydroxyl radicals (54,158) to bind to several Ter sites. It bound extremely avidly to the TerB site; the Tus-TerB complex had a measured dissociation constant (K D ) of 3.4 ϫ 10 Ϫ13 M and a dissociation half-life in vitro of 550 min at pH 7.5 in a buffer containing 150 mM potassium glutamate (54).…”

Section: The Tus Gene and Tus Proteinmentioning

confidence: 99%

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

Neylon

Kralicek

Hill

et al. 2005

Microbiol Mol Biol Rev

134

149

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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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