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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...

No abstract

Replication forks are arrested at specific sequences to facilitate a variety of DNA transactions. Forks also stall at sites of DNA damage, and the regression of stalled forks without rescue can cause genetic instability. Therefore, unraveling the mechanisms of fork arrest and of rescue of stalled forks is of considerable general interest. In Schizosaccharomyces pombe, products of two matingtype switching genes, swi1 and swi3, participate in fork arrest at the mating-type switch locus. Here, we show that these proteins also act at three termini (Ter) also called replication fork barriers in the spacer regions of rDNA but not at a fourth site, RFP4, which is nonfunctional when present in a plasmid. Two of the Swi1p-and Swi3p-dependent sites were also dependent on the transcription terminator Reb1p. Furthermore, hydroxyurea-induced replication stress mimicked the effect of swi1 or swi3 mutations at these sites. A swi1 mutant that failed to arrest forks at the mating-type fork barrier RTS1 was functional at the rDNA Ter sites, suggesting some specificity of action. Both WT and mutant forms of Swi1p were physically localized at the Ter sites in vivo. The results support the notion that Swi1p and Swi3p act at several different protein-DNA complexes in the rDNA spacer regions to arrest replication but that not all fork barriers required their activity to arrest forks.mating-type switching genes ͉ genome stability ͉ replication termination

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...

Unlike the chromosome of Escherichia coli that needs only one replication initiator protein (origin recognition protein) called DnaA, many plasmid replicons require dual initiators: host-encoded DnaA and a plasmidencoded origin recognition protein, which is believed to be the major determinant of replication control. Hitherto, the relative mechanistic roles of dual initiators in DNA replication were unclear. Here, we present the first evidence that DnaA communicates with the plasmid-encoded π initiator of R6K and contacts the latter at a specific N-terminal region. Without this specific contact, productive unwinding of plasmid ori γ and replication is abrogated. The results also show that DnaA performs different roles in host and plasmid replication as revealed by the finding that the ATP-activated form of DnaA, while indispensable for oriC replication, was not required for R6K replication. We have analyzed the accessory role of the DNA bending protein, integration host factor (IHF), in promoting initiator-origin interaction and have found that IHF significantly enhances the binding of DnaA to its cognate site. Collectively, the results further advance our understanding of replication initiation.

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We have isolated mutants of the pi initiator protein of the plasmid R6K that are defective in DNA looping in vitro but retain their normal DNA binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. One such looping defective mutant called R6 was determined to be a proline to leucine change at position 46 near the N terminus of the pi protein. Using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the distantly located alpha and beta origins, we show that the looping defective initiator protein fails to activate the alpha and beta origins but derepresses initiation from the normally silent gamma origin in vivo. The results conclusively prove that DNA looping is required to activate distant replication origins located at distances of up to 3 kb from the replication enhancer.