An investigation of RNR regulation in fission yeast by confocal laser scanning FRET and near-TIRF microscopy

Abstract

For genome integrity, adequate levels of deoxyribonucleotide (dNTPs) are essential to maintain faithful DNA replication and repair via the regulation of ribonucleotide reductase (RNR). In the fission yeast, RNR is composed of two subunits: Cdc22 and Suc22. The importance of Spd1 (RNR inhibitor) in Cdc22-Suc22 complex formation has been demonstrated by imaging of S. pombe containing fluorescent protein (FP) modified RNR subunit proteins in the presence of Spd1 and absence of Spd1 cells using confocal laser scanning microscopy. To investigate further the significant role of Spd1 in the regulation of RNR, 41 mutants created by Nestoras group. We used fluorescence resonance energy transfer (FRET) by acceptor photobleaching to investigate the RNR subunit interaction and provide evidence for a new model for the role of Spd1 in RNR regulation. Different treatments such as HU, 4NQO and heat shock have been used to investigate the effect of radical scavenging on the inhibition of RNR activity and induced DNA damage on S. pombe cell viability to elucidate further the role of Spd1 in the regulation of RNR. Finally a novel imaging technique, near-total internal reflection microscopy has been developed and applied with dual-view detection. The technique has been applied to image, simultaneously, the donor CFP and acceptor YFP channels of the FP-tagged RNR complex in the wild-type S. pombe cells and perform FRET measurements that are consistent with the confocal fluorescence results. In conclusion, a new hypothesis for the role of Spd1 has been drawn from the results, which is that the inhibitory role of Spd1 mediates the Suc22-Cdc22 (R1-R2) interaction to form a FRET competent but immature and inactive RNR complex, while with Spd1 deleted RNR is clearly active in a conformation that lacks FRET