DNA topoisomerase and supercoil accumulation across yeast genome

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).DNA拓扑异构酶（DNA topoisomerases）通过调控超螺旋动态变化，辅助DNA复制与转录事件的进行。本研究分析了酵母全基因组的超螺旋分布情况，并特别针对S期样本，对比了RNA聚合酶II（RNA pol2）与DNA拓扑异构酶的富集特征。研究数据显示，Top2与Hmo1可通过稳定可变DNA结构，维持基因边界处的负超螺旋状态。为解析DNA超螺旋张力在全基因组范围内的积累机制，我们将此前报道的方法[Naughton C等，2013]应用于出芽酵母体系：该体系中生物素分子通过连接子与胸苷一磷酸（TMP）结合，得到生物素化TMP（bTMP）。蛋白质的染色质免疫沉淀芯片（ChIP-chip）分析参照文献(Bermejo R等，2009)所述流程完成。针对RNA-DNA杂交体的DNA-RNA免疫沉淀芯片（DRIP-chip）实验则沿用此前报道的方法(Chan YA等，2014)开展。最终将检测得到的超螺旋区域与RNA聚合酶II（RNA pol2，RPB3-chip）、DNA拓扑异构酶（Top1-chip）及RNA-DNA杂交体（DRIP-chip）的富集结果进行对比分析。