Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the GJ/S boundary by using either cultures synchronized with a factor or Cdc-mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob)-), which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob)-) mutation cannot bypass all events in G1 phase because it fails to suppress temperaturesensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.A fundamental issue in cell biology is the control of the eukaryotic cell cycle. Alterations in cell cycle regulation are implicated in neoplastic cell growth and aberrations in early development (26,28,31), indicating that cell cycle control is a key contributor to understanding the basis of cancer and morphogenesis. One approach to understanding the regulation of the cell cycle involves the study of the cell division cycle (CDC) genes of the budding yeast Saccharomyces cerevisiae. Many CDC genes encode proteins which mediate unique and essential roles in the cell cycle.

The product of the CDC7 gene of Saccharomyces cerevisiae appears to have multiple roles in cellular physiology. It is required for the initiation of mitotic DNA synthesis. While it is not required for the initiation of meiotic DNA replication, it is necessary for genetic recombination during meiosis and for the formation of ascospores. It has also been implicated in an error-prone DNA repair pathway. Plasmids Use of the budding yeast Saccharomyces cerevisiae as a model for studies on the eucaryotic cell cycle relies heavily on temperature-sensitive mutations in cell division cycle (CDC) genes (32, 35). Characterization of these mutants has led to the formulation of a model in which progression through the cell cycle is determined by a set of interrelated pathways, each organized as a dependent sequence of events requiring the action of specific gene products. One such pathway, operating late in the Gi phase of the cell cycle, requires the function of the CDC7 gene product (12). Cells carrying a thermosensitive lesion in the CDC7 gene arrest at the restrictive temperature as budded cells with separated spindle-pole bodies but without an elongated spindle apparatus and without initiating DNA synthesis (5, 12). Upon return to permissive conditions cdc7 cells are able to enter the S phase and subsequently complete a round of DNA synthesis without further protein synthesis (14).In contrast to the requirement for CDC7 function to initiate mitotic DNA synthesis, premeiotic DNA replication occurs normally in cdc7 homozygous diploids under the restrictive condition (43). However, these diploids fail to form a synaptonemal complex, to show commitment to genetic recombination, or to form ascospores (40). Thus, although cdc7 strains are defective in both mitotic and meiotic cell cycles, the lesion appears to affect each pathway in a quite distinct manner. In addition to having roles in the mitotic and meiotic pathways, the CDC7 gene product has been implicated in DNA repair as a member of the RAD6 epistasis group, since strains carrying a cdc7 mutation show almost no mutagenic repair in response to a variety of damaging agents (31).To elucidate the role of the CDC7 gene product in the various cellular functions in which it is implicated and to determine whether differential expression of the CDC7 gene is involved with its cell cycle functions, we and others (24) have begun a molecular analysis of the CDC7 gene. In this paper we describe the cloning of the CDC7 gene, the characterization of its transcriptional product, the nucleotide sequence of the gene, and the regions of homology between the predicted protein products of the CDC7 and CDC28 genes. MATERIALS AND METHODSStrains and media. Escherichia coli HB101 (F-thi leu pro hsdR hsdM recA end!) and HW87 [F-A(araD139-leu) lacX74 galK hsdR rpsL srb recA] were used as hosts for the routine maintenance and propagation of plasmids. Bacterial cultures were grown in L broth or supplemented M9 medium (26); when necessary, ampicillin was added to media to a final concentration of...

Over the past 40 years, the search for new antibiotics has been largely restricted to well-known compound classes active against a standard set of drug targets. Although many effective compounds have been discovered, insufficient chemical variability has been generated to prevent a serious escalation in clinical resistance. Recent advances in genomics have provided an opportunity to expand the range of potential drug targets and have facilitated a fundamental shift from direct antimicrobial screening programs toward rational target-based strategies. The application of genome-based technologies such as expression profiling and proteomics will lead to further changes in the drug discovery paradigm by combining the strengths and advantages of both screening strategies in a single program.

The interaction of Hoechst 33258, a fluorescent DNA stain, has been studied by using the synthetic, self-complementary oligonucleotide duplex d(CGCGAATTCGCG)2. Spectrofluorometric Scatchard analysis indicated that there was only a single class of binding site and that the 1:1 complex had a dissociation constant of (3.47 +/- 0.1) X 10(-6) M at 25 degrees C. Spectroscopic titration by high-field 1H NMR confirmed the 1:1 complex and by means of ID and 2D (NOESY, COSY) techniques the binding site was defined as the minor groove formed by the AATT stretch. Plentiful cross-peaks were measurable and resonance doubling occurred because of the lifting of the diad symmetry of the oligonucleotide on ligand binding. Many individual resonances of both strands of the DNA could be assigned for the complex because of these features, along with the occurrence of slow exchange on the NMR time scale. The results of this NMR spectroscopic solution study were compared with those of previous X-ray crystallographic studies of the same complex. From nuclear Overhauser effect data measured for the complex, a detailed three-dimensional model was constructed with the aid of molecular graphics.

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