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.本研究旨在探究常用核酸酶处理方案及其二价金属阳离子辅因子对天然延伸转录本测序(NET-seq)实验中聚合酶I(Pol I)占有率的影响。此前已有研究团队在针对聚合酶II(Pol II)占有率的NET-seq实验方案中，使用了微球菌核酸酶(MNase)+氯化钙(CaCl2)以及脱氧核糖核酸酶I(DNase I)+氯化锰(MnCl2)的处理方式，但此类处理对聚合酶占有率的影响尚未得到充分探究。为此，本研究针对四种处理方案——氯化钙(CaCl2)、氯化钙+微球菌核酸酶(CaCl2+MNase)、氯化锰(MnCl2)以及氯化锰+脱氧核糖核酸酶I(MnCl2+DNase I)——在体内对聚合酶I(Pol I)占有率的影响展开了检测。结果显示，将聚合酶I暴露于氯化钙或氯化锰环境中，均会使其在核糖体DNA(rDNA)模板上的占有率出现显著下降；单独使用核酸酶（微球菌核酸酶或脱氧核糖核酸酶I）处理，并不会在氯化钙或氯化锰单独处理的基础上，对体内聚合酶I的占有率产生额外的显著影响。进一步分析发现，相较于氯化钙处理，氯化锰处理对聚合酶I占有率的抑制效果更为显著，这提示在体内环境中，氯化锰相较氯化钙可更高效地激活聚合酶I的内在切割活性。综上，本研究结果表明，在针对聚合酶I的NET-seq实验方案中加入此类处理方式（尤其是二价金属阳离子处理步骤），会显著改变聚合酶的占有率。