Helena Nedergaard Nielsen:
Aspects of Ribonucleotide Reductase Regulation and Genome Stability

Date: 14-03-2014    Supervisor: Christian Holmberg

In all living cells, synthesis of the DNA building blocks, deoxyribonucleoside triphosphates (dNTPs), is tightly regulated to ensure a precise DNA replication to maintain genomic stability. Ribonucleotide reductase (RNR) is the enzyme responsible for reducing ribonucleotides to their deoxy forms. RNR consists of two subunits: R1 and R2, both of which are essential. The activity of RNR is strictly regulated to control the dNTP pool, both by allosteric feedback control and transcriptional and translational controls. Four inhibitory proteins of RNR have been identified in yeast: Spd1 in fission yeast, and Sml1, Hug1, and Dif1 in budding yeast. An elevated, as well as a reduced dNTP pool is shown to lead to an increase in spontaneous mutation rates, hence regulation of RNR is very important in order to maintain genomic stability. No human inhibitory proteins have yet been identified to regulate the human RNR enzyme.

In this study regulation of human RNR was investigated using a fission yeast strain that depended solely on the human genes of R1 and R2 for dNTP synthesis. Even though this strain could grow like wild-type fission yeast it was hypersensitive to hydroxyurea (HU) and depended on the checkpoint protein Rad3. Furthermore, strains with the human RNR genes showed defects in meiosis, which was partially rescued by yeast R1, suggesting a role for yeast RNR during meiosis besides synthesizing dNTPs. In this study a human cDNA library was also created, which can be expressed in fission yeast. This library can be used in complementation screens by human genes in fission yeast.