Article 15

Hennessy, K.M., A. Lee, E. Chen, & D. Botstein (1991) A group of interacting yeast DNA replication genes. Genes Dev. 5: 958-969.

The authors of this article make very elegant use of genetic analysis to explore the function of a new series of essential genes involved in the initiation of DNA synthesis. Strains carrying cold-sensitive mutations at two loci, CDC45 and CDC54, arrest growth at low temperatures with a phenotype similar to cdc7 mutants; that is, they arrest as large budded cells with a single nucleus (Moir et al., 1982). Mutations that suppress the cold-sensitive phenotype of cdc45 and cdc54 alleles were isolated in CDC46 (MCM5, BOB1) and CDC47 and strains carrying only these suppressor mutations were found to exhibit a temperature-sensitive phenotype and arrested with the same phenotype as the cold-sensitive cdc45 and cdc54 mutants at the nonpermissive temperature (Moir et al., 1982).

The finding that strains carrying mutations in any one of these genes exhibit an apparently identical phenotype that is similar to the cdc7 mutant phenotype strongly suggests that they all participate in the same essential function, a step required for the initiation of DNA synthesis. The cold-sensitive phenotype of some of the mutant alleles also suggests that the encoded proteins might be components of a large multimeric protein complex. This suggestion is further supported by the results presented in this article.

1. Describe how Southern analysis of CHEF gel-separated chromosomes is used here to map a gene to a particular chromosome.

2. Table 1 lists the various alleles of CDC45, CDC46, CDC47, and CDC54 used in this analysis and their growth phenotype, cold sensitive or temperature sensitive. The genetic interactions among these genes are summarized in Figure 1. Diagram the crosses demonstrating the following genetic interactions. For each give the genotype and phenotype of the parent strains, the diploid strain, and the spores of a parental ditype, tetratype, and nonparental ditype tetrads.

(a) CDC46 and CDC45 exhibit allele-specific suppression. (Requires two crosses.)

(b) CDC46 and CDC54 show allele-specific synthetic lethality.

3. Mutant strains carrying cdc45-l, cdc46-l, and cdc47-l cdc45-\ double mutants all arrest at the same point of initiation of DNA replication at the non-permissive temperature. How was this demonstrated? Do cdc7 and dbf4 temperature-sensitive mutants exhibit this same phenotype? Be sure to reference the article from this case study series in which this was demonstrated.

4. Studies of the mechanism of recombination indicate that homologous DNA exchange events initiate at sites of double-strand breaks and single-stranded regions of chromosomes. Suggest a hypothesis explaining why cdc46-l, cdc45-1, and cdc54-l mutant strains might exhibit increased rates of recombination. Is your hypothesis consistent with the studies of chromosome integrity described in Figure 4? Explain.

5. PEST sequences are regions of a protein rich in proline, aspartate, glutamate, serine, and threonine.

(a) What is believed to be the role of PEST sequences in proteins?

(b) Why do the authors find it so interesting that several of these Cdc proteins contain PEST sequences?

6. What does the term 'immunologically related' mean and how is it demonstrated?

7. What is the significance of finding a mouse protein that is immunologically related to Cdc46p, particularly given the role of Cdc46p in a process common to all eukaryotic cells—the initiation of DNA replication?

REFERENCE

Moir, D., S.E. Stewart, B.C. Osmund, & D. Botstein (1982) Cold-sensitive cell division cycle mutants of yeast: isolation, properties, and pseudoreversion studies. Genetics 100: 547-563.

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