Genetic analysis of the fission yeast

Abstract: ABSTRACT Ribonucleotide reductase (RNR) catalyzes the rate‐limiting step of dNTP production. The availability of finely tuned dNTP pools is very important to maintain genome stability during DNA synthesis for replication and repair. Therefore the activity of RNR is tightly regulated via various mechanisms. In yeast species RNR inhibitors proteins prevent the RNR activation in the G1 and G2 phases. Upon entry into S phase or in response to DNA damage where the DNA synthesis is required, these proteins are degraded through distinct mechanisms. Comparative genomic studies in fission yeast identified the spd2 gene. The amino acid sequence of the Spd2 protein shows high similarity to the previously characterized RNR inhibitor protein, Spd1. Spd2 protein shows homology to Spd1 in distinct domains that are proven to be important for the function and regulation of Spd1. Using site directed mutagenesis I disrupted these domains in Spd2. I used two different assays to analyse the phenotypes corresponding to these mutations. In the first assay, I screened for the ability of these mutation to rescue the growth of Δddb1 strains in the absence of rad3 gene, a key component of DNA structure‐dependent checkpoint. In the second assay I looked at the ability of these mutations to restore meiosis and spore formation in Δddb1 strains. The Ddb1 is a component of CRL4Cdt2 E3 ubiquitin ligase that is involved in the degradation of RNR inhibitor proteins in fission yeast. Furthermore, I looked at the stability of different Spd2 mutant proteins. Mutations in the PIP degron of the Spd2 were able to restore growth in strains that lack both active proteasome and checkpoint. Mutations in the PIP degron were able to restore meiosis and spore formation in Δddb1 strains. Result of the SDS gel, suggest that the PIP degron is important for the stability of the protein. Mutations in the PIP degron and Helix II made the protein less stable compared to the mutations in the C terminus. From these results I conclude that the PIP degron of the Spd2 is important for its function and regulation. Popular science summary: Another guardian for the genome Cell cycle is the essential mechanism for reproduction and it takes place in all living cells. It is an orderly sequence of events in which a cell duplicates its contents and then divides into two. In general, the cell cycle is divided into four different phases: G1 phase, S phase, G2 phase and M phase. DNA synthesis, replication and chromosome duplication occurs during the S phase. Four deoxyribonucleotides (dNTPs), the building blocks of the DNA are required for DNA synthesis in S phase or in response to DNA damage. In majority of organisms, the dNTPs are generated by the enzyme ribonucleotide reductase (RNR). Optimal supply of dNTPs during replication and repair is vital and imbalanced dNTP pools are mutagenic and can lead to genome instability and cancer. For this reason the activity of RNR is tightly regulated by several mechanisms. In yeast species a special group of inhibitor proteins inhibit the RNR activity when DNA synthesis is not required. Degradation of these proteins via is necessary for RNR activation. In fission yeast (Schizosaccharomycec pombe) such a protein, Spd1 is identified and well characterized. The Spd1 is ubiquitinated and degraded via the function of CRL4Cdt2 E3 ligase (composed of the Cul4, DDB1, and the DCAF subunit Cdt2). Recently, via the comparative genomic studies in S. pombe, spd2 gene was identified. The Spd2 protein sequence shows high similarity to Spd1. Preliminary studies suggests that Spd2 also is a potential RNR inhibitor and might function in the same pathway as the Spd1. The aim of this study is to characterize the function of different domains that are similar between Spd1 and Spd2 and proved to be important for the function and regulation of Spd1 protein. Using oligonucleotide mutagenesis, mutations in these domains are generated and two informative assays are used to analyze the role of these domains in Spd2 function in Δddb1 strains. These cells cannot degrade the inhibitor proteins cannot replicate their DNA and their survival extremely depends on the function DNA damage checkpoints, RAD proteins. In the first assay, I screened for the ability of these mutation to rescue the growth of cells that lack ddb1 and rad3. In the second assay I looked at the ability of these mutations to restore meiosis and spore formation in Δddb1 strains. These cells cannot proceed through the meiosis hence they are not able to produce spores. Furthermore, I looked at the stability of different Spd2 mutant proteins. Mutations in the PIP degron of the Spd2 were able to restore growth in strains that lack both active proteasome and checkpoint. Mutations in the PIP degron were able to restore meiosis and spore formation in Δddb1 strains. Result of the SDS gel, suggest that the PIP degron is important for the stability of the protein. Mutations in the PIP degron and Helix II made the protein less stable compared to the mutations in the C terminus. From these results I conclude that the PIP degron of the Spd2 is important for its function and regulation. Many cancer cells have constitutively high RNR activity and it will be interesting to learn whether RNR inhibitor proteins regulate dNTP pools in other systems like human cells. Advisor: Professor Jure Piskur, Professor Olaf Nielsen, Master´s Degree project 60 credit in Molecular Genetics 2012 Department of Biology Lund University, Department of Biology University of Copenhagen Subject of the degree project: Genetic analysis of the Fission yeast Spd2 protein