The regulation of the replication of the genome is critical in the basic process of cell division. The genetic material must be duplicated correctly, completely, and at the appropriate time during the cell division cycle. I use the single-celled organism Schizosaccharomyces pombe, or fission yeast, as a model for the study of eukaryotic DNA replication.
The fission yeast mutant, cdc24-M38, was originally identified in the cell division cycle (cdc) mutant screen twenty years ago by Kim Nasmyth and Paul Nurse as a mutant defective in the DNA synthesis phase, or S phase, of the cell cycle. Loss of cdc24 function resulted in the arrest of the mutant cells with a single nucleus and an apparently replicated genome. During my postdoctoral work with Susan Forsburg and in collaboration with Kathy Gould at Vanderbilt University, we cloned the gene, cdc24+, and demonstrated it is an essential fission yeast gene encoding a novel protein with no obvious homologs in the genome databases. Analysis of the chromosomes from mutant cdc24 strains by pulsed field gel electrophoresis demonstrated that loss of cdc24 function also resulted in chromosome breakage, which is not characteristic of S phase arrest mutants. I identified a new fission yeast gene, dna2+, a putative DNA helicase, which suppresses the temperature sensitive growth phenotype of the cdc24 mutant strains.
In my laboratory, the focus of my research is to define the role of Cdc24p in genome maintenance. My work has indicated that this mutant has genetic interactions with a group of proteins involved not only with DNA replication but also with DNA repair. These interactions present the exciting possibility that this gene may have a functional significance in the development of diseases of genome stability. I have demonstrated that not only the DNA helicase, Dna2p, but also the flap endonuclease Rad2p, homologous to the human FEN-1, suppress the cdc24 mutant growth defect at the restrictive temperature. This is an intriguing suite of suppressors since they are not only involved with resolution of the lagging strand intermediates (Okazaki fragment maturation), but also required for DNA repair. Thus, identification of these suppressors suggests that Cdc24p may have a role in both DNA replication and DNA repair.
Overall, my approach to defining the function of Cdc24p in the fission yeast cell cycle utilizes genetics, molecular biology, cell biology, and biochemistry approaches. For undergraduate student projects, I am proposing several projects, which provide a useful introduction to the biology of fission yeast, encompass an initially genetics-based approach, and require the students to learn molecular biological techniques. These experiments provide flexibility for the student in terms of time management, since at almost any point, yeast strains can be stored in the refrigerator or freezer. Furthermore, the successful identification of new suppressors will generate a collection of potential projects for additional students.
I.Isolation of high copy suppressors of cdc24-M81 mutant allele
I have already identified two suppressors of the cdc24 mutant by suppressing the temperature sensitive phenotype of the cdc24-M38 allele. However, while this mutant allele results in the carboxy terminal truncation of Cdc24p (wild type Cdc24p, 501 amino acid polypeptide; mutant Cdc24p-M38, 370 amino acid polypeptide), the distinct cdc24-M81 allele carries a point mutation (serine to proline at amino acid 136). High copy suppression of the cdc24-M81 strain will likely recover the same genes that suppress cdc24-M38 but may identify novel suppressors which are specific to the lesion of the cdc24-M81 allele or fail to suppress cdc24-M38 because Cdc24p-M38 is truncated. I have available two fission yeast genomic libraries and a cDNA library for this high copy suppression screen. Plasmids that suppress the cdc24 temperature sensitive defect can be easily recovered from the yeast strains and analyzed by standard molecular biological techniques.
II.Isolation of second site suppressors of cdc24 mutant alleles
This strategy to identify genomic suppressors of the cdc24 lesion will define genes that interact directly with Cdc24p or perhaps bypass cdc24 function. This approach requires mutagenesis of each cdc24 mutant strain (cdc24-M38 and cdc24-M81) followed by recovery at the restrictive temperature. We will employ two kinds of mutagenesis in this screen: a chemical mutagen (nitrosoguanidine) and ultraviolet light. cdc24 mutants which are now able to form colonies at the restrictive temperature will be subjected to further analysis to determine whether the cdc24 mutation has reverted or a second site mutation in a suppressor gene ?X? has compensated for the cdc24 mutation. Genomic and cDNA libraries are available to clone the suppressor gene. If the suppressor has a conditional phenotype in the absence of the cdc24 mutation, then the suppressor gene can be isolated by rescue of its phenotype. However, if the suppressor gene does not have a conditional phenotype, it can be recovered in the cdc24 background by identifying plasmids that restore the cdc24 temperature sensitivity.
III.Generation of a conditional allele of dna2+
My unpublished work indicates that dna2+ is an essential gene in fission yeast. The generation of temperature sensitive mutants of any essential gene is a standard procedure in yeast molecular genetics. The protocol requires a diploid that is heterozygous null for dna2+ (that is, dna2+/?dna2::his3+. I have constructed this strain) and a wild type dna2+ gene on a plasmid. Treatment of the plasmid DNA with the chemical mutagen hydroxylamine results in a mutagenized plasmid DNA library. This collection of mutagenized dna2 plasmids is transformed into the diploid strain, and haploid strains containing the disrupted dna2 allele and the mutant dna2 plasmid are recovered at room temperature. This scheme requires that the plasmid encoded dna2 gene be functional at room temperature. These strains are then assayed for temperature sensitivity to identify conditional alleles of the dna2. Isolation of a conditional allele of dna2 will enable further characterization of the gene by providing a means to analyze the physiological consequences of the loss of dna2 function, to identify genetic interactions with cdc24 and other S phase mutants, and to isolate suppressors of the dna2 lesion.
Although Cdc24p is a novel protein, it genetically interacts with replication proteins conserved in evolution: an essential replicative DNA helicase and a flap endonuclease potentially involved with the development of cancer and trinucleotide repeat diseases. This initial approach will generate a genetic framework for determining cdc24 function and may provide insight into diseases of genome instability.