title: 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 G(1) 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 G(1) cells and highly induced in damaged S/G(2) 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.
authors.affiliation: Department of Biological Engineering and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
authors.forename: Aprotim
authors.initials: A
authors.lastname: Mazumder
authors.forename: Katja
authors.initials: K
authors.lastname: Tummler
authors.forename: Mark
authors.initials: M
authors.lastname: Bathe
authors.forename: Leona D
authors.initials: LD
authors.lastname: Samson
mesh_terms: D002453:Cell Cycle; D004249:DNA Damage; D005455:Fluorescent Antibody Technique / Q000379:methods; D015966:Gene Expression Regulation, Fungal; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000032:analysis / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D059010:Single-Cell Analysis / Q000379:methods*; D014158:Transcription, Genetic
chemical_list: D021122:Protein Subunits; D012333:RNA, Messenger; D012264:Ribonucleotide Reductases