Testing the usage of divalent metal cations and nucleases during NET-seq for Pol I

The goal of this study was to determine the effects of commonly used nuclease treatments and their divalent metal cation cofactors on Pol I occupancy during NET-seq. Other groups have previously used micrococcal nuclease (MNase) + CaCl2 and deoxyribonuclease I (DNase I) + MnCl2 in NET-seq protocols probing for Pol II occupancy. However, the effect of these treatments on polymerase occupancy has not been fully explored. Therefore, we tested the impact of these treatments (CaCl2, CaCl2 + MNase, MnCl2, and MnCl2 + DNase I) on Pol I occupancy in vivo. We found that the exposure of Pol I to either CaCl2 or MnCl2 caused a significant reduction in occupancy on the rDNA template. Nuclease treatment (with either MNase or DNase I) did not cause an additional significant effect on Pol I occupancy in vivo beyond that of either CaCl2 or MnCl2. Finally, we found that MnCl2 treatment caused a more significant reduction in Pol I occupancy as compared to CaCl2, suggesting that perhaps MnCl2 stimulates the intrinsic cleavage activity of Pol I more robustly vs. CaCl2 in vivo. Altogether, these findings indicate that the inclusion of these treatments (especially that of divalent metal cations) in NET-seq protocols for Pol I cause a significant change in the polymerase occupancy. NET-seq was performed in triplicate with a WT yeast strain across five different treatment conditions: (1) Untreated Control, (2) CaCl2 Treated, (3) CaCl2 + MNase Treated, (4) MnCl2 Treated, and (5) MnCl2 + DNase I Treated.

本研究旨在探究常用核酸酶处理方法及其二价金属阳离子辅因子对新生转录本测序（NET-seq）实验中RNA聚合酶I（Pol I）结合占有率的影响。此前已有研究团队在针对RNA聚合酶II（Pol II）结合占有率的NET-seq实验方案中，采用了微球菌核酸酶（MNase）+氯化钙（CaCl₂）以及脱氧核糖核酸酶I（DNase I）+氯化锰（MnCl₂）的处理组合，但上述处理方式对聚合酶结合占有率的影响尚未得到充分探索。
据此，本研究针对四种处理方案（氯化钙、氯化钙+MNase、氯化锰、氯化锰+DNase I），检测了其在体内对Pol I结合占有率的影响。研究发现，将Pol I暴露于CaCl₂或MnCl₂环境中，均会使其在核糖体DNA（rDNA）模板上的结合占有率出现显著降低。仅采用核酸酶处理（无论使用MNase还是DNase I），相较于单独使用CaCl₂或MnCl₂，并未对体内的Pol I结合占有率造成额外的显著影响。
进一步分析显示，相较于CaCl₂处理，MnCl₂处理对Pol I结合占有率的抑制作用更为显著，这提示在体内条件下，MnCl₂或许比CaCl₂更能有效激活Pol I的内在切割活性。综上，本研究结果表明，在针对Pol I的NET-seq实验方案中加入上述处理方式（尤其是二价金属阳离子处理），会对聚合酶的结合占有率造成显著改变。
本研究针对五种不同处理条件下的野生型酵母菌株开展了三次重复NET-seq实验，五种处理条件分别为：(1) 未处理对照组，(2) CaCl₂处理组，(3) CaCl₂ + MNase处理组，(4) MnCl₂处理组，(5) MnCl₂ + DNase I处理组。