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...

Although DNA looping between the initiator binding sites (iterons) of the replication origin (ori) of a plasmid and the iterons located in a cis-acting control sequence called inc has been postulated to promote negative control of plasmid DNA replication, not only was definitive evidence for such looping lacking, but also the detailed molecular mechanism of this control had not been elucidated. Here, we present direct evidence showing that both the monomeric and the dimeric forms of the RepE initiator protein of F factor together promote pairing of incC-oriF sites by DNA looping. By using a reconstituted replication system consisting of 26 purified proteins, we show further that the DNA loop formation negatively regulates plasmid replication by inhibiting the formation of an open complex at the replication origin, thus elucidating a key step of replication control.

We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori ␥ of R6⌲, elongation of the forks, and their termination at specific replication terminators. The initiation was strictly dependent on the plasmid-encoded initiator protein and on the hostencoded initiator DnaA. The wild type was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication. The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase. The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication. In contrast, wild type -mediated replication in vivo requires IHF. Using a template that contained ori ␥ flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products. A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo. Many features of replication in the reconstituted system appeared to mimic those of in vivo replication. The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.

Jacob, Brenner, and Cuzin pioneered the development of the F plasmid as a model system to study replication control, and these investigations led to the development of the "replicon model" (Jacob, F., Brenner, S., and Cuzin, F. (1964) Cold Spring Harbor Symp. Quant. Biol. 28, 329 -348). To elucidate further the mechanism of initiation of replication of this plasmid and its control, we have reconstituted its replication in vitro with 21 purified host-encoded proteins and the plasmid-encoded initiator RepE. The replication in vitro was specifically initiated at the F ori (oriV) and required both the bacterial initiator protein DnaA and the plasmidencoded initiator RepE. The wild type dimeric RepE was inactive in catalyzing replication, whereas a monomeric mutant form called RepE* (R118P) was capable of catalyzing vigorous replication. The replication topology was mostly of the Cairns form, and the fork movement was unidirectional and mostly from right to left. The replication was dependent on the HU protein, and the structurally and functionally related DNA bending protein IHF could not efficiently substitute for HU. The priming was dependent on DnaG primase. Many of the characteristics of the in vitro replication closely mimicked those of in vivo replication. We believe that the in vitro system should be very useful in unraveling the mechanism of replication initiation and its control.

The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1⌬ strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the ⌱407⌻ mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.

The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein but also at a distance, caused by -mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476 -45484). Although the in vitro reconstituted system promotes ori ␥-specific initiation of replication by a mutant form of the initiator called *, the wild type (WT) is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT . The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that is recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.

A typical plasmid replicon of Escherichia coli, such as ori ␥ of R6K, contains tandem iterons (iterated initiator protein binding sites), an AT-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein DnaA, and a binding site for the DNA-bending protein IHF. R6K also contains two structurally atypical origins called ␣ and ␤ that are located on either side of ␥ and contain a single and a half-iteron, respectively. Individually, these sites do not bind to initiator protein but access it by DNA looping-mediated interaction with the seven -bound ␥ iterons. The protein exists in 2 interconvertible forms: inert dimers and active monomers. Initiator dimers generally function as negative regulators of replication by promoting iteron pairing ("handcuffing") between pairs of replicons that turn off both origins. Contrary to this existing paradigm, here we show that both the dimeric and the monomeric are necessary for ori ␣-driven plasmid maintenance. Furthermore, efficient looping interaction between ␣ and ␥ or between 2 ␥ iterons in vitro also required both forms of . Why does ␣-␥ iteron pairing promote ␣ activation rather than repression? We show that a weak, transitory ␣-␥ interaction at the iteron pairs was essential for ␣-driven plasmid maintenance. Swapping the ␣ iteron with one of ␥ without changing the original sequence context that caused enhanced looping in vitro caused a significant inhibition of ␣-mediated plasmid maintenance. Therefore, the affinity of ␣ iteron for -bound ␥ and not the sequence context determined whether the origin was activated or repressed.

In the ribosomal DNA (rDNA) array of Saccharomyces cerevisiae, DNA replication is arrested by the Fob1 protein in a site-specific manner that stimulates homologous recombination. The silent information regulator Sir2, which is loaded at the replication arrest sites by Fob1, suppresses this recombination event. A plasmid containing Fob1-binding sites, when propagated in a yeast strain lacking SIR2 is integrated into the yeast chromosome in a FOB1-dependent manner. We show that addition of nicotinamide (NAM) to the culture medium can stimulate such plasmid integration in the presence of SIR2. Pulsed-field gel electrophoresis analysis showed that plasmid integration occurred into chromosome XII. NAM-induced plasmid integration was dependent on FOB1 and on the homologous recombination gene RAD52. As NAM inhibits several sirtuins, we examined plasmid integration in yeast strains containing deletions of various sirtuin genes and observed that plasmid integration occurred only in the absence of SIR2, but not in the absence of other histone deacetylases. In the absence of PNC1 that metabolizes NAM, a reduced concentration of NAM was required to induce plasmid integration in comparison with that required in wild-type cells. This study suggests that NAD metabolism and intracellular NAM concentrations are important in Fob1-mediated rDNA recombination.