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. U nrepaired DNA damage can result in cell growth arrest, apoptosis, premature aging, neurodegeneration, and cancer (1, 2). Because most DNA repair pathways require de novo synthesis of DNA, damaged DNA signals the increased production and activation of the ribonucleotide reductase (RNR) enzyme (3-5). In almost all eukaryotes, the functional RNR enzyme consists of a large and a small subunit (3). The Saccharomyces cerevisiae genes RNR1 and RNR3 code for the large-subunit proteins, while RNR2 and RNR4 code for the small-subunit proteins (Fig. 1). The active form of the small subunit is an Rnr2-Rnr4 heterodimer (6, 7), and it relocalizes to the cytoplasm from the nucleus upon DNA damage (4, 8) to make the functional holoenzyme with the large subunit. Additionally, upon DNA damage, the transcription of all RNR genes is induced by the Mec1-Rad53 pathway (9, 10), which also controls the subcellular localization of the Rnr2-Rnr4 heterodimer (11) and the activation of the RNR enzyme (12, 13). Much of our understanding of the response of RNR to DNA damage as a function of cell cycle stage comes from bulk biochemical studies involving the model eukaryote S. cerevisiae (Fig. 1) (14-16). However, the synchronization methods employed in these studies may alter normal cell behavior. Further, mean values probed in bulk population studies mask information on cell-tocell variability in response, which is clearly resolvable with singlecell-level imaging (17)(18)(19)(20). Moreover, mRNA and protein levels and localization are usually measured in separate experiments, and few studies have explored the measurement of both gene products...

Yeast Proteome Dynamics from Single Cell Imaging and Automated Analysis

“…Additional studies have examined the collection in various conditions and several have quantified abundance of the GFP-tagged proteins (Benanti et al, 2007;Breker et al, 2013;Mazumder et al, 2013), but all have relied on manual assessment of localization, which is non-quantitative and suboptimal since assignments made by different individuals can be subject to biases and often show poor agreement (for review, see Chong et al, 2012).…”

Cell cycle–dependent spatial segregation of telomerase from sites of DNA damage

“…To test this possibility, another cytological marker of DSB, γ-H2A, was combined with FISH on TLC1 RNA. In contrast to Rfa1-GFP, γ-H2A form several foci in mammalian and yeast cells after DNA damage ( Rogakou et al, 1999 ; Mazumder et al, 2013 ). In yeast, γ-H2A accumulation is a direct readout of Mec1 activity at a DSB ( Ira et al, 2004 ).…”

Measuring cell cycle-dependent DNA damage responses and p53 regulation on a cell-by-cell basis from image analysis

“…Single transcript detection has been described before [26][27][28]. In brief, the cells were grown on glass-bottom plates in Full Medium and fixed with 4% PFA in PBS.…”

“…Such bulk biochemistry-based techniques also cannot report on cell to cell variability of DDR or subcellular localization of gene products nor do they yield information about possible correlations between two measured responses on a cell-by-cell basis [23,24]. Flow cytometry does report on the cell-to-cell variability in a population of cells [25] but lacks localization information and cannot be combined with the methods which yield absolute transcript counts like singlemolecule RNA fluorescence in situ hybridization (smFISH) [26][27][28]. To overcome these limitations we report a microscopy-based technique to study the cell cycle dependence of DDR in asynchronous cells in culture.…”