Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Mohanty, Bidyut K.; Sahoo, Trilochan; Bastia, Deepak

doi:10.1074/jbc.273.5.3051

Cited by 34 publications

(36 citation statements)

References 53 publications

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“…Although Tus protein is able to arrest DnaB, simian virus 40 Tag (17,18), and several RNA polymerases (10,11), it has some helicase specificity, as shown by its inability to arrest helicases involved in rolling circle replication and DNA repair and conjugative transfer (19).…”

Section: Discussionmentioning

confidence: 99%

Mechanism of termination of DNA replication of Escherichia coli involves helicase–contrahelicase interaction

Mulugu

Potnis

Shamsuzzaman

et al. 2001

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

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Section: Discussionmentioning

confidence: 99%

Mechanism of termination of DNA replication of Escherichia coli involves helicase–contrahelicase interaction

Mulugu

Potnis

Shamsuzzaman

et al. 2001

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

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“…This is in contrast with prokaryotic replication terminator proteins Tus of Escherichia coli and RTP of Bacillus subtilis, which arrest not only the cognate replicative helicase (e.g., DnaB of E. coli) by binding to the Ter sites and with the replicative helicase (43) but also a variety of other helicases, such as simian virus 40 tumor antigen (SV40 Tag) (2,8) and PriA, but not helicases involved in rolling circle replication, chromosome transfer (helicase I), or DNA repair (e.g., UvrD and helicase II) (25,26,50). These terminator proteins also arrest RNA polymerase of E. coli with the same polarity as that for DnaB (38). Mutant forms of the terminator protein that interact with Ter DNA with normal or near-normal affinity but fail to interact with the helicases also fail to arrest forks in vivo and in vitro, thereby supporting the conclusion that in addition to terminator protein-Ter interaction, protein-protein interaction with the helicase is critical to the mechanism (33,43).…”

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Mechanistic Insights into Replication Termination as Revealed by Investigations of the Reb1-Ter3 Complex of Schizosaccharomyces pombe

Biswas

Bastia

2008

Molecular and Cellular Biology

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Relatively little is known about the interaction of eukaryotic replication terminator proteins with the cognate termini and the replication termination mechanism. Here, we report a biochemical analysis of the interaction of the Reb1 terminator protein of Schizosaccharomyces pombe, which binds to the Ter3 site present in the nontranscribed spacers of ribosomal DNA, located in chromosome III. We show that Reb1 is a dimeric protein and that the N-terminal dimerization domain of the protein is dispensable for replication termination. Unlike its mammalian counterpart Ttf1, Reb1 did not need an accessory protein to bind to Ter3. The two myb/SANT domains and an adjacent, N-terminal 154-amino-acid-long segment (called the myb-associated domain) were both necessary and sufficient for optimal DNA binding in vitro and fork arrest in vivo. The protein and its binding site Ter3 were unable to arrest forks initiated in vivo from ars of Saccharomyces cerevisiae in the cell milieu of the latter despite the facts that the protein retained the proper affinity of binding, was located in vivo at the Ter site, and apparently was not displaced by the "sweepase" Rrm3. These observations suggest that replication fork arrest is not an intrinsic property of the Reb1-Ter3 complex.

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“…Second, the Tus-Ter complex also arrests RNA polymerase of E. coli in a polar mode at or near the coordinates Ϫ6 and Ϫ11, immediately upstream of the blocking face of Tus-Ter complex (13,18) (see Fig. 1B).…”

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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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Mechanistic Studies on the Impact of Transcription on Sequence-specific Termination of DNA Replication and Vice Versa

Cited by 34 publications

References 53 publications

Mechanism of termination of DNA replication of Escherichia coli involves helicase–contrahelicase interaction

Mechanism of termination of DNA replication of Escherichia coli involves helicase–contrahelicase interaction

Mechanistic Insights into Replication Termination as Revealed by Investigations of the Reb1-Ter3 Complex of Schizosaccharomyces pombe

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

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