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).

Lack of G1/S control destabilizes the yeast genome via replication stress-induced DSBs and illegitimate recombination

“…2D). This methodology enables access to the DNA structures in chromosomal samples (Krol et al, 2017;Lewinska et al, 2014). In the swi6Δ-derived samples, we observed replication bubbles and Ψ-type structures (the latter formed during fork regression).…”

“…To gain insight into aberrant DNA structures developed in chromosomes during DSB repair, which might accumulate with hampered repair, we performed a chromosome comet assay (Lewinska et al, 2014;Adamczyk et al, 2016;Krol et al, 2017). We analyzed a band representing two chromosomes, VII and XV, cut from the gel after PFGE.…”

“…Yeast chromosome integrity was analyzed as described previously (Krol et al, 2017). Yeast cells were embedded in 20-μl plugs of low-melting-point InCert agarose (Lonza, Basel, Switzerland) and digested with Zymolyase 100T (BioShop, Burlington, ON, Canada), and then with proteinase K (Sigma-Aldrich) and RNase A at 30°C with gentle rotation (4 r.p.m.)…”

“…Aberrant DNA structures in the chromosomal bands excised from the PFGE gel were visualized via a chromosome comet assay (see Fig. S7 for graphical description of principles of the method and some examples of assay results), as described in Krol et al (2017). In short, the chromosomal bands were placed distally on poly-L-lysine-coated microscopic slides (CometSlide, Trevigen, Gaithersburg, MD) and shielded with 40 μl of 0.6% New Sieve low-melting-point agarose (Conda).…”

Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism

“…Yeast chromosome integrity was analyzed as described previously [168] with certain modifications. Yeast cells grown at 23°C were embedded in 20-μl plugs of low-melting-point SeaKem Gold agarose (Lonza, Basel, Switzerland).…”