Ribosomal DNA status inferred from DNA cloud assays and mass spectrometry identification of agarose-squeezed proteins interacting with chromatin (ASPIC-MS)
| Autor: | Marek Skoneczny, Anna Kurlandzka, Kamil Krol, Michal Dadlez, Justyna Jendrysek, Adrianna Skoneczna, Joanna Kaminska, Janusz Debski |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
DNA repair DNA-binding protein Biology DNA Ribosomal Genome Mass Spectrometry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine DNA structures Tandem repeat Humans nucleolus Gene Ribosomal DNA Genetics Sepharose Chromosome genome instability Chromatin Las17 030104 developmental biology Oncology chemistry 030217 neurology & neurosurgery DNA Research Paper |
| Zdroj: | Oncotarget |
| ISSN: | 1949-2553 |
| Popis: | // Kamil Krol 1 , Justyna Jendrysek 1 , Janusz Debski 2 , Marek Skoneczny 3 , Anna Kurlandzka 3 , Joanna Kaminska 3 , Michal Dadlez 2 , Adrianna Skoneczna 1 1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Warsaw, 02-106, Poland 2 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Mass Spectrometry Laboratory, Warsaw, 02-106, Poland 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Genetics, Warsaw, 02-106, Poland Correspondence to: Adrianna Skoneczna, email: ada@ibb.waw.pl Keywords: nucleolus, genome instability, DNA structures, DNA-binding protein, Las17 Received: May 16, 2016 Accepted: January 23, 2017 Published: February 15, 2017 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). |
| Databáze: | OpenAIRE |
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