Ribonucleotide reductase and DNA damage
Abstract: A prerequisite for a multicellular organism to survive is the ability to correctly replicate and repair DNA while minimizing the number of heritable mutations. To achieve this, cells need a balanced supply of deoxyribonucleoside triphosphates (dNTPs), the precursors for DNA synthesis. The rate-limiting step in de novo biosynthesis of dNTPs is catalyzed by the enzyme ribonucleotide reductase (RNR).The classic eukaryotic RNR enzyme consists of a large and a small subunit. Together, these subunits form a heterotetrameric RNR complex. The larger subunit harbours active sites whereas the smaller subunit contains a stable tyrosyl free radical. Both subunits are required for RNR activity.Since failure to correctly regulate de novo dNTP biosynthesis can lead to misincorporation of nucleotides into DNA, genetic abnormalities and cell death, RNR activity is tightly regulated. The regulation of RNR activity involves cell cycle-specific expression and degradation of the RNR proteins, as well as binding of allosteric effectors to the large RNR subunit.In this thesis, in vitro assays based on purified recombinant RNR proteins, in combination with in vivo assays, have been used successfully to study the regulation of RNR activity in response to DNA damage. I present new findings regarding the function of an alternative mammalian RNR small subunit, and on the role of a small RNR inhibitor protein of fission yeast, during normal growth and after DNA damage. I also show conclusively that there are fundamental differences in the regulation of dNTP biosynthesis between the cells of higher and lower eukaryotes after DNA damage.
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