title: Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS).
abstract: Ribosomal RNA-encoding genes (rDNA) are the most abundant genes in eukaryotic genomes. To meet the high demand for rRNA, rDNA genes are present in multiple tandem repeats clustered on a single or several chromosomes and are vastly transcribed. To facilitate intensive transcription and prevent rDNA destabilization, the rDNA-encoding portion of the chromosome is confined in the nucleolus. However, the rDNA region is susceptible to recombination and DNA damage, accumulating mutations, rearrangements and atypical DNA structures. Various sophisticated techniques have been applied to detect these abnormalities. Here, we present a simple method for the evaluation of the activity and integrity of an rDNA region called a "DNA cloud assay". We verified the efficacy of this method using yeast mutants lacking genes important for nucleolus function and maintenance (RAD52, SGS1, RRM3, PIF1, FOB1 and RPA12). The DNA cloud assay permits the evaluation of nucleolus status and is compatible with downstream analyses, such as the chromosome comet assay to identify DNA structures present in the cloud and mass spectrometry of agarose squeezed proteins (ASPIC-MS) to detect nucleolar DNA-bound proteins, including Las17, the homolog of human Wiskott-Aldrich Syndrome Protein (WASP).
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Warsaw, 02-106, Poland.
authors.forename: Kamil
authors.initials: K
authors.lastname: Krol
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Warsaw, 02-106, Poland.
authors.forename: Justyna
authors.initials: J
authors.lastname: Jendrysek
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Mass Spectrometry Laboratory, Warsaw, 02-106, Poland.
authors.forename: Janusz
authors.initials: J
authors.lastname: Debski
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Genetics, Warsaw, 02-106, Poland.
authors.forename: Marek
authors.initials: M
authors.lastname: Skoneczny
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Genetics, Warsaw, 02-106, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kurlandzka
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Genetics, Warsaw, 02-106, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Kaminska
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Mass Spectrometry Laboratory, Warsaw, 02-106, Poland.
authors.forename: Michal
authors.initials: M
authors.lastname: Dadlez
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Warsaw, 02-106, Poland.
authors.forename: Adrianna
authors.initials: A
authors.lastname: Skoneczna
mesh_terms: D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D012685:Sepharose
chemical_list: D002843:Chromatin; D004275:DNA, Ribosomal; D012685:Sepharose
keywords: DNA structures; DNA-binding protein; Las17; genome instability; nucleolus