Yeast nucleolar chromatin during transcription, UV-DNA damage transcription arrest, and nucleotide excision repair: insights from proteomes of purified rRNA genes (12/12)
Description
The protein composition of rRNA genes in chromatin was examined before irradiation (-UV), immediately after irradiation, and during NER at various post-UV time points. A yeast strain was genetically engineered to carry each chromosomal 35S-rRNA gene tagged with LexA DNA-binding sites and flanked by and R-recombinase recognition sequences. Additionally, the yeast strains were expressing recombinant LexA fused to a tandem affinity purification (TAP) tag and the R-recombinase from Z. rouxii under the control of an inducible promoter. This strain was named “35S-RS”. After inducing R-recombinase expression, 35S-rRNA genes were released from the chromosome as circular 35S-rRNA gene chromatin rings. Cells were directly harvested or irradiated with UVC and incubated for different times (0, 10, 20, 30, 60 and 120 min) in growth medium to let DNA repair to happen before harvesting. After cell lysis, 35S-rRNA gene rings were purified via the TAP-tagged LexA protein in buffers containing either 100 or 200 mM KCl. The protein composition of the affinity purified 35S-rRNA gene chromatin rings was determined through HPLC-MS/MS using a Dionex Ultimate 3000 nanoHPLC system (Dionex Corporation) coupled to an OrbiTrap QExactive mass spectrometer (ThermoFisher Scientific). As a control, a yeast strain in which the chromosomal 35S-rRNA genes were neither tagged with LexA DNA-binding sites nor flanked by R-recombinase recognition sequences but also expressed recombinant LexA-TAP and the R-recombinase from Z. rouxii were treated and processed in parallel. This strain was named “Control”. The data presented here are the raw data from the mass spectrometer. Data analyses were done and are presented in the related research paper. A Microsoft Excel file is added to present which file corresponds to which experiment and how the data were grouped to run MaxQuant analyses. It also contains information about the nomenclature of samples used to analyze the MaxQuant output files with the LFQ-analyst platform (https://analyst-suites.org/). Additionally, the FASTA document used to process the raw data on MaxQuant is joined. It contains the Uniprot Saccharomyces cerevisiae (strain ATCC 204508 / S288c) database (2019, 6049 entries), complemented with the LexA-TAP protein and the R recombinase (R. rouxii) sequences. Part 12 of 12 : dataset from "Yeast nucleolar chromatin during transcription, UV-DNA damage transcription arrest, and nucleotide excision repair: insights from proteomes of purified rRNA genes" Proteomics raw data from rRNA genes purified with 200 mM KCl right after UV irradiation (60 min UV + 120 min UV)