Effects Of Deregulation Of Ribonucleotide Reductase In Mice
Nucleotide levels play a critical role in genome stability, and proper regulation of these pools is crucial for survival. Ribonucleotide reductase (RNR) catalyzes the rate- limiting step in de novo dNTP biosynthesis. The enzyme is composed of two non-identical homodimeric subunits, a large subunit encoded by a single gene Rrm1, and a small subunit encoded by either Rrm2 or a DNAdamage-inducible gene p53R2. In mammals, RNR activity is thought to be controlled by two main mechanisms: limitation of small subunit protein levels and allosteric feedback control. Studies in yeast and cultured mammalian cells have shown that disabling RNR regulation results in increased mutation rates. The objective of the studies described here was to develop mouse models to elucidate the physiological consequences of disrupting RNR regulatory mechanisms in vivo, either individually or in combination. We first overrode the control of RNR protein levels by generating multiple mouse strains that featured overexpression of individual RNR subunits. Mice that overexpressed either small RNR subunit developed lung cancer at a high frequency, while mice that overexpressed both Rrm1 and either small RNR subunit developed age-dependent mitochondrial DNA depletion in addition to lung cancer. These findings highlight the impact of RNR deregulation on the stability of both the nuclear and mitochondrial DNA genomes. In order to disrupt RNR allosteric control, we also generated transgenic mice that overexpress a feedbackresistant form of Rrm1, Rrm1-D57N, as well as knock-in mice in which the D57N mutation was introduced directly into the Rrm1 genomic locus. These mouse strains were used individually or in various combinations to assess the effects of loss of RNR regulation on genome maintenance, tumorigenesis, and survival. Although mice that overexpress Rrm1-D57N were grossly normal, mice that overexpress Rrm1-D57N in combination with either small RNR subunit were inviable, indicating that simultaneous loss of both main RNR regulatory mechanisms is incompatible with survival. Preliminary work suggested large dNTP pool alterations in the skeletal muscle of bitransgenic neonates, resulting in pathological changes in multiple organ systems and premature lethality. It was also found that mice homozygous for the Rrm1D57N knock-in mutation are inviable, further indicating that proper allosteric feedback control of RNR is critical for survival. All together, these results indicate that RNR plays a central role in genome maintenance, and that alterations in RNR activity are detrimental for survival.
ribonucleotide reductase; nucleotide metabolism; DNA replication
Weiss, Robert S.
Schimenti, John C.; Liu, Jun; Stover, Patrick J
Ph.D. of Genetics
Doctor of Philosophy
Dissertation or Thesis