In vivo structure-mediated regulation of ribonucleotide reductase in S. pombe

Doctoral thesis English OPEN
Schreurs, Ann-Sofie (2012)
  • Subject: QD0415 | QP0603.R52

Sufficient and balanced pools of deoxyribonucleotide triphophates (dNTPs) is\ud crucial for high-fidelity DNA replication as well as correct DNA repair. The enzyme\ud RiboNucleotide Reductase (RNR) catalyses NDP to dNDP and is therefore an\ud essential enzyme by providing the “building blocks” to the cells. dNTPs production\ud needs to be tightly regulated in order to minimize mutation frequencies and prevent\ud genome instability.\ud \ud RNR in S. pombe is composed of two proteins, Cdc22R1 and Suc22R2, and has\ud been described as a heterotetramer with a dimer of each subunit: the big subunit\ud Cdc22R1 and the small subunit Suc22R2. S. pombe also posseses an RNR inhibitor:\ud Spd1, as well as a second RNR regulator Spd2 which has been newly discovered.\ud Spd1 has been demonstrated to inhibit RNR and to regulate its activity throughout the\ud cell cycle. The detailed mechanism of the RNR regulation during the cell cycle or after\ud DNA damage is not entirely clear, as are the means of inhibition by Spd1. In order to\ud shed some light on the RNR complex and its regulation, we used various microscopybased\ud methods to study RNR in vivo as well as in vitro.\ud \ud The data of this thesis suggest there are different forms of active RNR\ud heterocomplexes, found throughout the cell cycle in the cytoplasm as well as in the\ud nucleus. We propose that the precise stoichiometry of subunits in the complexes may\ud vary, or that the complex conformation may be modified in an Spd1-dependent\ud manner. In addition, treatment of the cells with a UV mimetic agent, 4NQO, seems to\ud promote RNR regulation in an Spd1-dependent manner. On the contrary, inhibition of\ud RNR by HydroxyUrea (HU) affects the RNR in a possible structure-related manner,\ud independently of Spd1 or Spd2. The in vivo observations correlate with structural\ud and/or oligomerization modifications of the RNR, representing a novel RNR regulation\ud in S. pombe.
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