9308014 Merrill In building yeast, virtually of the genes encoding proteins involved in DNA replication contain multiple copies of the sequence ofAOGCGTNA in their 5' regions. This sequence, termed a Mlu1 cell cycle box or MCB, confers late G1 activity on heterologous promoters and specifically binds a complex called DSC1 in band shift assays. One components component of DSC1 is Swi6. In Aswi6 antibodies binds DSC1 in band shift assays. An as yet unidentified protein in DSC1 is thought to be the primary DNA recognition polypeptide. Swi6 is also present in another complex that binds a related sequence, CACGAAAA, termed in SCB element, that occurs upstream from the HO and G1 cyclin genes. In the SCB complex, a second protein, Swi4, is thought to be the primary DNA recognition polypeptide. Given the sequence similarity of MCBs and SCBs and the fact that both complexes contain Swi6, it is likely that the DNA recognition polypeptide of the MCB complex resembles Swi4 In preliminary work several yeast and human genes involved in regulating DNA synthesis gene expression were isolated from yeast expression vector libraries using a genetic screen based on activation of reporter genes containing an MCB element upstream activation sequence. One of the novel yeast genes, termed DSA3, for DNA synthesis gene activator 3, is a particularly strong activator. DSA3 may encode the protein that directly recognizes MCB elements or may encode an upstream regulator of the MCB-binding complex. During the proposed 1-year project period, the role of DSA3 in controlling late G1 DNA synthesis gene expression will be investigated as follows. The primary structure of the activator will be completed to see if it contains domains homologous to regions of other known proteins. The function of the activator will be investigated by determining the effect of deleting the activator gene on cell viability, DSC1 activity, and DNA synthesis gene periodicity; by assaying the MCB binding activ ity of the activator protein when if is produced by in vitro transcription and translation; and by identifying proteins that physically interact with the activator protein. Results of the analysis will set the stage for future studies designed to identify other components of the MCB element activation mechanism, and to determine whether similar DNA synthesis gene activators are operative in higher eucaryotes. %%% The gene encoding enzymes required for genome replication are turned on shortly before DNA synthesis begins, Human tumor suppressor proteins have been implicated in DNA synthesis gene regulation, but the overall regulatory mechanism is unresolved. Studies in yeast have contributed greatly to our understanding of how these genes are regulated, but two important question remain unanswered: 1) what protein specifically recognizes the DNA synthesis genes and targets them f or activation; 2) what protein senses that the cell has decided to replicated its genome and triggers the DNA synthesis gene activation mechanism. Using a genetic screen, a novel gene that strongly and specifically activates yeast DNA synthesis genes has been isolated. During the proposed project period, the novel gene will be sequenced and its gene targeting protein or triggering protein. ***
