DNA topoisomerase and supercoil accumulation across yeast genome. 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). 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△

DNA拓扑异构酶（DNA topoisomerase）通过调控超螺旋变化，协助DNA复制与转录过程。本研究对酿酒酵母基因组中的超螺旋分布情况进行了分析，并特别针对S期，将其与RNA聚合酶Ⅱ（RNA pol2）以及DNA拓扑异构酶的富集情况进行了对比。本研究数据显示，拓扑异构酶Ⅱ（Top2）与Hmo1可通过稳定非经典DNA结构，维持基因边界处的负超螺旋。为解析DNA超螺旋张力在基因组中的累积机制，我们将此前报道的方法[Naughton C等，2013]应用于酿酒酵母体系，该方法中生物素分子通过连接臂与胸苷单磷酸（TMP）结合，得到生物素化胸苷单磷酸（bTMP）。蛋白质的染色质免疫沉淀芯片（ChIP-chip）分析参照此前文献方法完成（Bermejo R等，2009）。针对RNA-DNA杂交链的DNA-RNA免疫沉淀芯片（DRIP-chip）实验参照已有方法进行（Chan YA等，2014）。随后将超螺旋区域与RNA聚合酶Ⅱ（RPB3-chip）、DNA拓扑异构酶（Top1-chip）以及RNA-DNA杂交链（DRIP-chip）的检测结果进行了比对。实验整体设计：本研究在以下酿酒酵母菌株中开展了蛋白质芯片（Protein-chip）、生物素化胸苷单磷酸芯片（bTMP-chip）以及DNA-RNA免疫沉淀芯片（DRIP-chip）实验：野生型（W303）、top2-1基因敲除株（top2-1△）、hmo1基因敲除株（hmo1△）、top1基因敲除株（top1△）、top2-1与hmo1双基因敲除株（top2-1;hmo1△）、top2-1与top1双基因敲除株（top2-1;top1△）、携带空载质粒的野生型菌株（WT-Ctrlplasmid）、携带TopA过表达质粒的野生型菌株（Wt-TopAplasmid）、携带空载质粒的top2-1;top1双基因敲除株（top2-1;top1△-Ctrlplasmid）、携带TopA过表达质粒的top2-1;top1双基因敲除株（top2-1;top1△-TopAplasmid）、sen1温度敏感突变株（sen1-cl）、rrm3基因敲除株（rrm3△）、rnh1基因敲除株（rnh1△）