Ribonucleotides embedded in genomic DNA of Aicardi-Goutières syndrome (AGS)-orthologous mutants of Saccharomyces cerevisiae using ribose-seq protocol
Ribonucleoside monophosphates (rNMPs) are abundantly found in DNA of cells, with over a million counts in the mammalian genome and thousands in budding yeast DNA. The embedded rNMPs alter DNA properties, increasing DNA fragility and mutability. Mutations in any of the three subunits of ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS orthologous mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics tool, we studied the rNMP incorporation frequency, distribution, composition, and patterns in these yeast mutants. We found significantly more abundant rNMPs in the nuclear genome of rnh201-G42S than in wild-type and rnh203-K46W-mutant cells. Moreover, we observed distinct sequence contexts and genomic sites with abundant rNMP incorporation (hotspots) in rnh201-G42S and rnh203-K46W cells, anticipating similar rNMP patterns in human DNA carrying the orthologous AGS mutations. Overall design: Strain BY4742, a derivative of S288C that was used in the S. cerevisiae gene disruption project, and have MATa mating type. Standard genetic and molecular biology methods were used for growth, gene disruption, isolation of mutants, selection, genome engineering, colony PCR, and sequence analysis of genomic DNA. The ribose-seq libraries derived from the wild-type RNase H2 or null-rnh201 genotype from S. cerevisiae cells containing the AGS mutations, affect RNase H2 activity on single rNMPs in DNA but allow cleavage at long RNA/DNA hybrids. We engineered two AGS orthologous mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W.  We constructed three or more ribose-seq libraries for each genotype using two or three different sets of restriction enzymes (RE1: DraI, EcoRV, and SspI; RE2: AluI, DraI, EcoRV, and SspI; and RE3:  HaeIII and RsaI). This strategy allowed us (i) to verify that the conclusions taken from our analyses of ribose-seq data are not influenced by a particular set of restriction enzymes used to fragment the DNA extracted from the different yeast species and genotypes, and (ii) to further confirm reproducibility of the results. After ribose-seq, the fastq files were trimmed and aligned to sacCer2 reference genome using Ribosemap toolkit to make the respective bam files available. Coordinate module of Ribose-Map and custom scripts were used to filter experimental artifacts and get the coordinates of rNMPs and converted to bedGraph format.