A large number of yeast genes, including several of the cyclins, are required for the transition between G1 and S. These genes are transcribed at two different times. One group of genes, including CLN3, SWI4, and CDC6 are turned on at the M/G1 boundary. The PI has identified a promoter element (ECB=early cell cycle box) necessary for expression of these genes at this time. The protein Mcm1p is one protein involved in activating transcription of genes with ECB boxes. Both C1n3p and Swi4p are required for the regulated expression of the second group of genes. These genes, including CLN1 and CLN2 (as well as many other genes), are expressed near the G1/S border. The genes of the second group require the promoter elements called SCBs and MCBs. These promoter elements are bound by two types of transcriptional complexes. One complex includes Swi4p and Swi6p, whereas the other includes Mbp1 and Swi6p. Most of the research proposal is an investigation of the proteins and protein binding sites required for these two waves of transcriptional activation. The first Specific Aim is to examine the G1/S-specific protein complexes as well as the promoter elements of the genes regulated at this point in the cell cycle. As described above, many of the promoters transcribed at the G1/S border contain sequences called SCB or MCB boxes that are bound by complexes containing Swi4p and Swi6p or Swi6p and Mbp1p. It has been shown, however, that the expression of the CLN1 and CLN2 genes is regulated by Swi 4p, although these genes do not have a canonical SCB or MCB box. Using Swi4p and Swi6p produced by in vitro translation, The PI will use site selection to identify the sequences recognized by the Swi4p/Swi6p complex. Antibodies directed against Swi6p will be used to purify the complexes. If these methods work for the Swi4p/Swi6p complex, she will use similar methods to examine the DNA binding properties of the Swi6p/Mbp1 complex. Sequences identified by these methods as binding sites will be fused to reporter genes for assays of in vivo activity. The PI will also attempt to localize the upstream DNA sequences of CLN1 and CLN2 that interact with the Swi4p complex. This localization will be done either by using band shifts and footprint analysis or by in vivo transcription assays. The second Specific Aim concerns the mechanism of G1/S-specific transcription. The Swi4p/Swi6p complex is bound to the SCB region throughout G1, but activation of transcription occurs only at the G1/S boundary. One model to explain the activation is that the Cln3p/Cdc28 kinases activates the Swi4p/Swi6p complex by phosphorylation. Although no such modifications affecting activity has been detected thus far, the level of the proteins in the cells is low and transient phosphorylation would be difficult to detect. The PI, therefore, will look for phosphorylation of the Swi4p/Swi6p complexes by Cln3p/Cdc28 kinases in vitro. Swi4p and Cln3p appear rate-limiting for G1 progression. The PI will determine whether constitutive expression of Swi4p and Cln3p changes the expression patterns of CLN1 and CLN2, as expected if these proteins are the only rate-limiting proteins. As described above, the SWI4, CLN3 and CDC6 genes are transcribed at the M/G1 border and the ECB element is required for this expression pattern. the Mcm1p is known to bind the ECB element. The third Specific Aim is to investigate the properties of this element and associated binding proteins in more detail. Fragments of the SWI4 and CLN3 genes that contain the ECB element will be fused to lacZ and HIS3 reporter genes. The response of the reporter to various growth regimens and various levels of the cyclin protein will be examined. The ECB element will be mutagenized in order to determine the sequences required for cell cycle- dependent expression. She will also examine the phenotypic consequences of deleting the promoter from the SWI4 and CLN3 genes. Two parameters that will be studied include cell size and DNA content, since the mutants may have an altered S-period or altered growth control. The CDC6 gene, which also contains an ECB element, encodes a protein required for the initiation of DNA replication. The PI will delete the ECB element from CDC6 and monitor the effect of this deletion on the length of the S-period and the fidelity of DNA replication. The DCD46 and DCD47 genes also have ECB boxes. It is thought that these genes might be involved in restricting DNA synthesis to one round of replication per cell cycle. The PI has shown that overexpression of Mcm1p apparently causes over-replication of DNA. She plans to determine whether this effect is mediated through CDC46 or CDC47 by determining whether the over-replication is eliminated in cdc46 and cdc47 mutants. It is likely that there are proteins other than Mcm1p involved in ECB- activated transcription. The PI proposes a number of genetic screens to identify such proteins. For example, she has found that overexpression of Mcm1p results in lethality. Therefore, she will identify suppressors of the lethality to detect interacting genes. Both recessive suppressors and high-copy suppressors will be isolated. The last Specific Aim is to investigate the checkpoint mechanism causing G1 arrest in response to DNA damage. She has previously shown that DNA damage results in a drop in CLN1 and CLN2 transcript levels. She has also shown that Swi6 becomes phosphorylated at novel positions within the protein in DNA-damaged cells in a Rad53p-dependent manner. The Rad53 kinase has been previously shown to be required for the operation of the checkpoint. Therefore, it is possible that DNA damage (by some unknown mechanism) activates the Rad53 kinase which inactivates (by phosphorylation) Swi6p, leading to diminished levels of Cln1 and Cln2, causing cell-cycle arrest. The PI will test whether purified Rad53p kinase phosphorylates the same peptides in Swi6p in vitro as she has observed in the in vivo experiments. If the same peptides are not phosphorylated in vitro, she will examine other relevant kinases (such as Mec1p). Finally, she will examine what cellular proteins are required to degrade Cln2p during the response to DNA damage. Cln2p degradation will be monitored in strains with mutations in various elements of the proteolytic machinery.
