Two independent pathways oJ transcriptional regulation that show functional homology have been identified in yeast. It has been demonstrated previously that SWI5 encodes a zinc finger DNA-binding protein whose transcription and cellular localization both are cell cycle regulated. We show that ACE2, whose zinc finger region is nearly identical to that of SWI5, shows patterns of cell cycle-regulated transcription and nuclear localization similar to those seen previously for SMTIS. Despite their similarities, SWI5 and ACE2 function in separate pathways of transcriptional regulation. SWI5 is a transcriptional activator of the HO endonuclease gene, whereas ACE2 is not. In contrast, ACE2 is a transcriptional activator of the CTSl gene (which encodes chitinase), whereas SWI5 is not. An additional parallel between the SWI5/HO pathway and the ACE2/CTS1 pathway is that HO and CTSl both are cell cycle regulated in the same way, and HO and CTSl both require the SWI4 and SWr6 transcriptional activators. Overproduction of either SWI5 or ACE2 permits transcriptional activation of the target gene from the other pathway, suggesting that the DNA-binding proteins are capable of binding in vivo to promoters that they do not usually activate. Chimeric SWI5/ACE2 protein fusion experiments suggest that promoter specificity resides in a domain distinct from the zinc finger domain.

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The Ace2p and Swi5p zinc finger proteins have nearly identical DNA-binding domains, yet in vivo they activate transcription of different genes, CTS1 and HO. We now demonstrate that Ace2p and Swi5p recognize sites in the CTS1 and HO promoters with the same affinities, raising the question of how promoter specificity is achieved by these proteins with similar DNA-binding domains. It has been previously shown that Swi5p binds to the HO promoter cooperatively with the Pho2p (Base2p/Grf10p) homeodomain protein, and we now show that Ace2p does not interact with Pho2p. Analysis of CTS1 promoter fragments inserted into a heterologous promoter identify a sequence 90 bp away from the Ace2p binding sites which is required to prevent activation by Swi5p through these binding sites. These results suggest that a regulatory protein bound to the CTS1 promoter is needed to prevent Swi5p from activating CT1S expression. A genetic screen was conducted to identify suppressor mutations which allow CTS1 expression in the absence of the Ace2p activator. The nce3 mutation suppresses the ace2 defect in CTS1 expression only if the strain contains a functional SWI5 gene, suggesting that NCE3 normally functions to prevent Swi5p from activating CTS1. The role of negative regulators such as NCE3, as well as the previously described SIN5 gene, in determining the promoter specificity of homologous activators is discussed.

A novel role for Rad53 in the initiation of DNA replication that is independent of checkpoint or deoxynucleotide regulation is proposed. Rad53 kinase is part of a signal transduction pathway involved in the DNA damage and replication checkpoints, while Cdc7-Dbf4 kinase (DDK) is important for the initiation of DNA replication. In addition to the known cdc7-rad53 synthetic lethality, rad53 mutations suppress mcm5-bob1, a mutation in the replicative MCM helicase that bypasses DDK's essential role. Rad53 kinase activity but neither checkpoint FHA domain is required. Conversely, Rad53 kinase can be activated without DDK. Rad53's role in replication is independent of both DNA and mitotic checkpoints because mutations in other checkpoint genes that act upstream or downstream of RAD53 or in the mitotic checkpoint do not exhibit these phenotypes. Because Rad53 binds an origin of replication mainly through its kinase domain and rad53 null mutants display a minichromosome loss phenotype, Rad53 is important in the initiation of DNA replication, as are DDK and Mcm2-7 proteins. This unique requirement for Rad53 can be suppressed by the deletion of the major histone H3/H4 gene pair, indicating that Rad53 may be regulating initiation by controlling histone protein levels and/or by affecting origin chromatin structure.

There is widespread agreement that the clamp loader of the Escherichia coli replicase has the composition DnaX3δδ’χψ. Two DnaX proteins exist in E. coli, full length τ and a truncated γ that is created by ribosomal frameshifting. τ binds DNA polymerase III tightly; γ does not. There is a controversy as to whether or not DNA polymerase III holoenzyme (Pol III HE) contains γ. A three-τ form of Pol III HE would contain three Pol IIIs. Proponents of the three-τ hypothesis have claimed that γ found in Pol III HE might be a proteolysis product of τ. To resolve this controversy, we constructed a strain that expressed only τ from a mutated chromosomal dnaX. γ containing a C-terminal biotinylation tag (γ-Ctag) was provided in trans at physiological levels from a plasmid. A 2000-fold purification of Pol III* (all Pol III HE subunits except β) from this strain contained one molecule of γ-Ctag per Pol III* assembly, indicating that the dominant form of Pol III* in cells is Pol III2τ2 γδδ’χψ. Revealing a role for γ in cells, mutants that express only τ display sensitivity to ultraviolet light and reduction in DNA Pol IV-dependent mutagenesis associated with double-strand-break repair, and impaired maintenance of an F’ episome.

Upon completion of synthesis of an Okazaki fragment, the lagging strand replicase must recycle to the next primer at the replication fork in under 0.1 second to sustain the physiological rate of DNA synthesis. We tested the collision model that posits that cycling is triggered by the polymerase encountering the 5′-end of the preceding Okazaki fragment. Probing with surface plasmon resonance, DNA polymerase III holoenzyme initiation complexes were formed on an immobilized gapped template. Initiation complexes exhibit a half-life of dissociation of approximately 15 minutes. Reduction of gap size to one nucleotide increased the rate of dissociation 2.5-fold and complete filling of the gap increased the off rate an additional three-fold (t½ ∼ 2 min). An exogenous primed template and ATP accelerated dissociation an additional four-fold in a reaction that required complete filling of the gap. Neither a 5′-triphosphate nor 5′-RNA terminated oligonucleotide downstream of the polymerase accelerated dissociation further. Thus, the rate of polymerase release upon gap completion and collision with a downstream Okazaki fragment is 1000-fold too slow to support an adequate rate of cycling and likely provides a backup mechanism to enable polymerase release when the other cycling signals are absent. Kinetic measurements indicate that addition of the last nucleotide to fill the gap is not the rate-limiting step for polymerase release and cycling. Modest (approximately 7 nucleotide) strand displacement is observed after the gap between model Okazaki fragments is filled. To determine the identity of the protein that senses gap filling to modulate affinity of the replicase for the template, we performed photo-crosslinking experiments with highly reactive and non-chemoselective diazirines. Only the α subunit cross-linked, indicating it serves as the sensor.

Examples of dynamic polymerase exchange have been previously characterized in model systems provided by coliphages T4 and T7. Using a dominant negative D403E polymerase (Pol) III ␣ that can form initiation complexes and sequester primer termini but not elongate, we investigated the possibility of exchange at the Escherichia coli replication fork on a rolling circle template. Unlike other systems, addition of polymerase alone did not lead to exchange. Only when D403E Pol III was bound to a -containing DnaX complex did exchange occur. In contrast, addition of Pol IV led to rapid exchange in the absence of bound DnaX complex. Examination of Pol III* with varying composition of or the alternative shorter dnaX translation product ␥ showed that -, 2 -, or 3 -DnaX complexes supported equivalent levels of synthesis, identical Okazaki fragment size, and gaps between fragments, possessed the ability to challenge pre-established replication forks, and displayed equivalent susceptibility to challenge by exogenous D403E Pol III*. These findings reveal that redundant interactions at the replication fork must stabilize complexes containing only one . Previously, it was thought that at least two s in the trimeric DnaX complex were required to couple the leading and lagging strand polymerases at the replication fork. Possible mechanisms of exchange are discussed.As with all cellular replicases, the DNA polymerase (Pol) 2 III holoenzyme (HE) of Escherichia coli is tripartite with a sliding clamp processivity factor (␤ 2 ), a clamp loader (DnaX complex, DnaX 3 ␦␦'), and an associated replicative polymerase (Pol III, ␣⑀) (for a review, see Ref. 2). In E. coli and many other bacteria, DnaX encodes two products: a shorter ␥ protein that has ATPase activity and the ability to support clamp loading on single-stranded DNA and a longer protein that has additional domains that interact with the DnaB 6 replicative helicase and Pol III. Cells that cannot express ␥ exhibit defects in UV viability and Pol IV-mediated mutagenesis (3). A proposal has been made that ␥ may be required to avoid the presence of a third Pol III at the replication fork that might outcompete other polymerases, such as Pol IV, that must enter the replication fork to resolve unrepairable lesions and to perform stress-induced mutagenesis functions (2, 3).An E. coli rolling circle replication system has been developed that exploits a 409-nt flapped circular template that has a 50:1 asymmetric GC content, allowing convenient distinction, quantification, and labeling of leading and lagging strands. The asymmetric GC distribution allows specific slowing of lagging strand synthesis by substitution of dGDPNP for dGTP. The V max for insertion of this analog is lower than the natural nucleotide, and the K m is higher, allowing "dialing in" a desired elongation rate without reducing the nucleotide to a level where its concentration is depleted during the progress of the reaction. Because the lagging strand half of the Pol III HE cycles when a new primer is made, even when the pre...

Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae. Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion in that the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4. In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase is shown to be an oligomer in the cell in this report. Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids. A molecular interaction between two different Cdc7 molecules was shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in approximately equal stoichiometry. Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein in that complexes of about 180 and 300 kDa were found. Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules.