Genome binding/occupancy profiling by genome tiling array
Summary
DNA topoisomerases assist DNA replication & transcription events by controlling supercoiling alterations. We investigated supercoil distribution across the yeast genome and compared with the accumulation of RNA pol2 and DNA topoisomerases particularly in S-phase. Our data indicate that Top2 along with Hmo1 maintain negative supercoil at gene boundaries by stabilizing alternative DNA structures. To understand how DNA superhelical tension accumulates across the genome we have adopted previously described method [Naughton C et al., 2013] to budding yeast where a biotin molecule was attached to TMP via a linker (bTMP). The Chip on chip analysis for proteins was carried out as described (Bermejo R et al., 2009). For RNA-DNA hybrids DRIP-chip is carried out as described previously (Chan YA et al., 2014). Supercoiled regions are then compared with RNA pol2 (RPB3-chip), DNA Topoisomerase (Top1-chip) & RNA-DNA hybrid (DRIP-chip).
Overall design
Protein-chip, bTMP-chip and DRIP-chip were carried out in following Saccharomyces Cerevisiae strains: wildtype (W303), top2-1△, hmo1△, top1△, top2-1;hmo1△, top2-1;top1△, WT-Ctrlplasmid, Wt-TopAplasmid, top2-1;top1△-Ctrlplasmid, top2-1;top1△-TopAplasmid, sen1-cl, rrm3△, rnh1△
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