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

Abstract

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...

The Escherichia coli Tus–Ter replication fork barrier causes site-specific DNA replication perturbation in yeast

“…The 'mousetrap' model of Tus-Ter RF pausing posits that a specific interaction is required between Tus and a critical 'locking cytosine' residue within TerB that is revealed by DNA strand separation 22 . An alternative model proposes that RF arrest at Tus-Ter is mediated by specific protein-protein interactions in E. coli 23,24 . Our data suggest that, although additional proteins may be required to reinforce RF pausing at Tus-Ter in E. coli 23,24 , these are clearly not required for Tus-Ter to function as a polar RF barrier in yeast.…”

“…An alternative model proposes that RF arrest at Tus-Ter is mediated by specific protein-protein interactions in E. coli 23,24 . Our data suggest that, although additional proteins may be required to reinforce RF pausing at Tus-Ter in E. coli 23,24 , these are clearly not required for Tus-Ter to function as a polar RF barrier in yeast. We therefore investigated the consequences of mutating the putative 'locking cytosine' residue within the TerB sites inserted adjacent to ARS305.…”

Site-Directed Mutagenesis and Yeast Reverse 2-Hybrid-Guided Selections to Investigate the Mechanism of Replication Termination

“…We confirmed that in such a substrate, DnaB sliding was arrested in a polar mode by the Tus-Ter complex only when present in the blocking orientation. These experiments led us to conclude that under physiological conditions a melting-flipping mechanism is not necessary (and probably does not occur) to cause polar fork arrest (Bastia et al, 2008).…”

“…We wished to rigorously test model 2, which postulated that DNA melting and base flipping together could explain polar fork arrest under a physiological salt concentration that permitted DNA replication to occur (Bastia et al, 2008). We reasoned that the model could be tested if one could temporally and spatially separate DNA unwinding by DnaB helicase from its ATP-dependent locomotion on DNA (double-or single-stranded).…”

Mitochondrial transcription

“…2a). 24 While it is possible that the unique conformation of MTERF1 on DNA might be responsible for the orientation dependence of the termination activity, it is tempting to speculate that perhaps interactions between MTERF1 and mitochondrial POLRMT, analogous to those observed between Tus and DnaB, might determine the polarity of termination events ( fig. 2b).…”