Termination of replication forks at the natural termini of the rDNA of Saccharomyces cerevisiae is controlled in a sequence-specific and polar mode by the interaction of the Fob1p replication terminator protein with the tandem Ter sites located in the nontranscribed spacers. Here we show, by both 2D gel analyses and chromatin immunoprecipitations (ChIP), that there exists a second level of global control mediated by the intra-S-phase checkpoint protein complex of Tof1p and Csm3p that protect stalled forks at Ter sites against the activity of the Rrm3p helicase (''sweepase''). The sweepase tends to release arrested forks presumably by the transient displacement of the Ter-bound Fob1p. Consistent with this mechanism, very few replication forks were arrested at the natural replication termini in the absence of the two checkpoint proteins. In the absence of the Rrm3p helicase, there was a slight enhancement of fork arrest at the Ter sites. Simultaneous deletions of the TOF1 (or CSM3), and the RRM3 genes restored fork arrest by removing both the fork-releasing and fork-protection activities. Other genes such as MRC1, WSS1, and PSY2 that are also involved in the MRC1 checkpoint pathway were not involved in this global control. This observation suggests that Tof1p-Csm3p function differently from MRC1 and the other above-mentioned genes. This mechanism is not restricted to the natural Ter sites but was also observed at fork arrest caused by the meeting of a replication fork with transcription approaching from the opposite direction.protein-protein interaction ͉ replication terminus ͉ terminator protein S ite-specific replication termini (Ter sites), also called replication fork barriers, are present in many prokaryotic chromosomes and at certain specific regions of eukaryotic chromosomes, such as the nontranscribed spacers of rDNA of Saccharomyces cerevisiae (Fig. 1A;. Binding of the cognate replication terminator proteins to the Ter sites causes programmed fork arrest that has special physiological functions (4). The terminator proteins of prokaryotes antagonize the activity of the replicative hexameric helicases in a polar mode (5-7), not only by binding to the Ter DNA but also by protein-protein contact with the replicative helicase (8, 9). Several aspects of the mechanism of fork arrest have been reviewed (4, 10).Eukaryotic replication termini are located in the nontranscribed spacers of rDNA of both budding and fission yeast (2,3,11,12). A protein called Fob1p is necessary for fork arrest at the tandem Ter sites present at the nontranscribed spacers of S. cerevisiae (13,14). A number of Ter-binding proteins have been discovered in Schizosaccharomyces pombe to date that bind to the replication termini located near the mating type locus and at the nontranscribed spacers of rDNA (15)(16)(17)(18)(19). Whether these proteins, like their analogues in prokaryotes, terminate replication by antagonizing the replicative helicase is not known at this time.In addition to the terminus-binding protein, two checkpoint proteins ca...

The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the single-stranded DNA or by a direct physical interaction between the DnaB and the DnaG proteins. In this paper we present evidence supporting direct interaction between the two proteins. We have mapped the surfaces of interaction on both DnaG and DnaB and show further that mutations that reduce the physical interaction also cause a significant reduction in primer synthesis. Thus, the physical interaction reported here appears to be physiologically significant.The multiprotein complex that initiates, elongates, and terminates DNA replication has been likened to a protein machine (1). The various component proteins of this machine have to work coordinately to successfully carry out the complex task of DNA replication. Thus, careful analyses of the interactions amongst the replisomal proteins are of critical importance for gaining further insights into the mechanism and control of the three steps of DNA replication. In both Escherichia coli and in mammalian cells, discontinuous synthesis of Okazaki fragments is primed by a class of enzymes called primases (2) that move along the length of the DNA template as a part of a multiprotein primosomal complex, synthesizing RNA primers at intermittent locations on the template DNA (3). Although a great deal of information has been uncovered regarding the enzymology of fork movement in prokaryotes and eukaryotes (3, 4), a number of significant questions still remain unanswered regarding the protein-protein interactions that control and drive fork movement.Studies on priming of phage single-stranded DNA (ssDNA), which is not coated with ssDNA binding protein, have shown that DnaB helicase (5) is needed along with DnaG primase for optimal primer synthesis (3). Two alternative mechanisms have been proposed to account for the need for DnaB in primer synthesis. One hypothesis was that DnaB interacted with ssDNA and created a conformation that allowed DnaG to load on to the DNA. This idea was based on the observation that DnaB binding induced a change in the secondary structure of ssDNA (5). The alternative hypothesis was that DnaB interacted directly with DnaG and with ssDNA, thus facilitating the loading of the primase on the template and͞or the activation of the enzyme (3). Although functional interaction between the C terminus of DnaG with DnaB was reported by Marians and coworkers (7,8), critical evidence showing physical interaction between the two protein has been elusive.To distinguish between the two alternative models of activation of the primase and͞or its loading on DNA, we have investigated the possible physical interaction between the helicase and the primase of Escherichia coli. In this paper we present evidence suppo...

FAM3C/ I nterleukin- l ike E MT I nducer (ILEI) is an oncogenic member of the FAM3 cytokine family and serves essential roles in both epithelial-mesenchymal transition (EMT) and breast cancer metastasis. ILEI expression levels are regulated through a non-canonical TGFβ signaling pathway by 3’-UTR-mediated translational silencing at the mRNA level by hnRNP E1. TGFβ stimulation or silencing of hnRNP E1 increases ILEI translation and induces an EMT program that correlates to enhanced invasion and migration. Recently, EMT has been linked to the formation of breast cancer stem cells (BCSCs) that confer both tumor cell heterogeneity as well as chemoresistant properties. Herein, we demonstrate that hnRNP E1 knockdown significantly shifts normal mammary epithelial cells to mesenchymal BCSCs in vitro and in vivo . We further validate that modulating ILEI protein levels results in the abrogation of these phenotypes, promoting further investigation into the unknown mechanism of ILEI signaling that drives tumor progression. We identify LIFR as the receptor for ILEI, which mediates signaling through STAT3 to drive both EMT and BCSC formation. Reduction of either ILEI or LIFR protein levels results in reduced tumor growth, fewer tumor initiating cells and reduced metastasis within the hnRNP E1 knock-down cell populations in vivo . These results reveal a novel ligand-receptor complex that drives the formation of BCSCs and represents a unique target for the development of metastatic breast cancer therapies.

Fob1p protein has been implicated in the termination of replication forks at the two tandem termini present in the non-transcribed spacer region located between the sequences encoding the 35 S and the 5 S RNAs of Saccharomyces cerevisiae. However, the biochemistry and mode of action of this protein were previously unknown. We have purified the Fob1p protein to near-homogeneity, and we developed a novel technique to show that it binds specifically to the Ter1 and Ter2 sequences. Interestingly, the two sequences share no detectable homology. We present two DNA replication in many prokaryotes is often terminated at sequence-specific replication termini (Ter sites). Replication terminator proteins specifically bind to the Ter sites and arrest forks in an orientation-specific mode with respect to the replication origins (1). In many eukaryotes, although every replicon in the multiorigin chromosomes does not have specific Ter sites, such sites are present in the nontranscribed spacer sequences in the rDNA from yeast to man (1, 2).In Escherichia coli and Bacillus subtilis, the Ter sites bind to replication termination proteins, called Tus and RTP, respectively, that are contrahelicases (3, 4) and impede replication fork movement not only by binding to the Ter sites but also by protein-protein interaction between the helicase and the terminator protein (5). The rDNAs of Saccharomyces cerevisiae (6, 7), Schizosaccharomyces pombe (8), Xenopus (9), mouse (10), pea (11), and humans (12) have similar replication fork arrest systems located in their nontranscribed spacer elements. In S. pombe, replication fork arrest has been shown to occur at and near the mating type switch locus (13-15).The rDNA locus of S. cerevisiae (Fig. 1) consists of 100 -200 tandem copies of 9.1-kb rDNA units present in chromosome number XII. Each unit of the rDNA consists of a 35 S rRNA and a 5 S rRNA gene that transcribe in opposite directions. A nontranscribed spacer called NTS2 containing an origin of replication called ARS 1 (autonomously replicating sequence) is located between the genes encoding the 35 S and the 5 S RNAs (6, 16).The two replication forks initiated at the ARS face unequal fate. The rightward moving fork moves through the 35 S rDNA in the same direction as that of transcription until it meets the fork coming from the opposite direction. But the leftward moving fork, after passing through the 5 S rRNA gene, is arrested at two Ter sites located in the nontranscribed spacer called NTS1 and is thus prevented from entering the 35 S rRNA gene. The two Ter sites (known as replication fork barrier or RFB sites) located in NTS1 arrest replication forks in an orientationdependent manner (6, 7). The Ter1 and Ter2 sites are separated from each other by a few nucleotides and can be further separated by inserting foreign DNA between them without affecting their activities (7).The Ter sites in prokaryotes and eukaryotes are recombinogenic (2). The yeast rDNA also contains a recombination hot spot HOT1 that consists of two elements called enhanc...

Replication forks are arrested at sequence-specific replication termini primarily, perhaps exclusively, by polar arrest of helicase-catalyzed DNA unwinding by the terminator protein. The mechanism of this arrest is of considerable interest. This paper presents experimental evidence in support of four major points pertaining to termination of DNA replication. First, the replication terminator proteins of both Escherichia coli and Bacillus subtilis are helicase-specific contrahelicases, i.e. the proteins specifically impede the activities of helicases that are involved in symmetric DNA replication but not of those involved in conjugative DNA transfer and rolling circle replication. Second, the terminator protein (Ter) of E. coli blocks not only helicase translocation but also authentic DNA unwinding. Third, the replication terminator protein of Gram-positive B. subtilis is a polar contrahelicase of the primosomal helicase PriA of Gram-negative E. coli. Finally, the blockage of PriA-catalyzed DNA unwinding was abrogated by the passage of an RNA transcript through the replication terminator protein-terminus complex. These results are significant because of their relevance to the mechanistic aspects of replication termination.