Single-Cell Analysis of Ribonucleotide Reductase Transcriptional and Translational Response to DNA Damage

Abstract

The ribonucleotide reductase (RNR) enzyme catalyzes an essential step in the production of deoxyribonucleotide triphosphates (dNTPs) in cells. Bulk biochemical measurements in synchronized Saccharomyces cerevisiae cells suggest that RNR mRNA production is maximal in late G₁ and S phases; however, damaged DNA induces RNR transcription throughout the cell cycle. But such en masse measurements reveal neither cell-to-cell heterogeneity in responses nor direct correlations between transcript and protein expression or localization in single cells which may be central to function. We overcame these limitations by simultaneous detection of single RNR transcripts and also Rnr proteins in the same individual asynchronous S. cerevisiae cells, with and without DNA damage by methyl methanesulfonate (MMS). Surprisingly, RNR subunit mRNA levels were comparably low in both damaged and undamaged G₁ cells and highly induced in damaged S/G₂ cells. Transcript numbers became correlated with both protein levels and localization only upon DNA damage in a cell cycle-dependent manner. Further, we showed that the differential RNR response to DNA damage correlated with variable Mec1 kinase activity in the cell cycle in single cells. The transcription of RNR genes was found to be noisy and non-Poissonian in nature. Our results provide vital insight into cell cycle-dependent RNR regulation under conditions of genotoxic stress.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01020-12.

ACKNOWLEDGMENTS

This work was supported by MIT Faculty Start-up Funds, the Samuel A. Goldblith Professorship, and a CEHS Pilot Project grant (deriving from NIH P30-ES002109) (to M.B., K.T., and A.M.), NIH R01-CA055042 and DP1-ES022576 (to L.D.S.), and the CSBi Merck-MIT fellowship (to A.M.).

We thank JoAnne Stubbe for the gift of Rnr antibodies and for insightful discussions. We thank Narendra Maheshri for insightful discussions, Arjun Raj for initial guidance with the FISH experiments, Katharina Ribbeck for access to the Zeiss Observer Z1 microscope, and Gerald Fink for the gift of the RC634 strain. The fluorophore-labeled DNA oligonucleotide probes were purified at the MIT Biopolymers facility.