Micrococcal nuclease does not substantially bias nucleosome mapping.
Micrococcal nuclease does not substantially bias nucleosome mapping.
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DOI：
10.1016/j.jmb.2012.01.043
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发表时间：
2012-03-30
影响因子：
5.6
通讯作者：
Keszenman-Pereyra, David
中科院分区：
文献类型：
--
作者：
Allan, James;Fraser, Ross M.;Owen-Hughes, Tom;Keszenman-Pereyra, David
关键词：
我们使用高通量测序在基因组 DNA 分子上绘制了序列定向核小体定位图。用鸡或蛙组蛋白重建制备的染色质，使用微球菌核酸酶 (MNase) 或半胱天冬酶激活的 DNase (CAD) 分别消化成单核小体。两种酶均优先切割核小体间（接头）DNA，尽管它们的机制明显不同。 MNase 迄今为止已被广泛用于核小体图谱绘制，尽管人们担心它可能会引入偏差。在确定了鸡或蛙组蛋白八聚体结合位点在每个 DNA 上的位置并定量了其强度后，使用多种标准对两种酶获得的结果进行了比较。两种酶在其首选切割位点都显示出序列特异性，尽管两种酶的这种选择性的性质是不同的。此外，由 CAD 核酸酶产生的核小体比用 MNase 产生的核小体长 8-10 bp，CAD 切割位点往往比相应的 MNase 切割位点距离核小体二元体更远 4-5 bp。尽管切割行为存在显着差异，但这两种核酸酶在每个测试的 DNA 上鉴定出基本上相同的核小体定位位点模式，这一观察结果与组蛋白类型无关。这些结果表明，在我们的条件下，使用 MNase 收集的核小体定位数据的偏差并不显着。 ► 我们使用两种不同的核酸酶测量核小体定位。 ► CAD 和 MNase 提供了等效的定位轮廓。 ► 结果与用于制备染色质的 DNA 和组蛋白类型无关。 ► 我们的数据与 MNase 提供有偏差的核小体定位测量的提议不一致。
We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8–10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4–5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant. ► We measured nucleosome positioning using two distinct nucleases. ► CAD and MNase provided equivalent positioning profiles. ► The results were independent of DNA and histone type used to prepare chromatin. ► Our data are not consistent with the proposal that MNase provides biased nucleosome positioning measurements.
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DOI：
10.1073/pnas.1003838107
发表时间：
2010-12-07
影响因子：
11.1
作者：
Locke, George;Tolkunov, Denis;Morozov, Alexandre V.
通讯作者：
Morozov, Alexandre V.
影响因子：
11.4
作者：
Segal, Eran;Widom, Jonathan
通讯作者：
Widom, Jonathan
影响因子：
64.5
作者：
Liu, XS;Zou, H;Wang, XD
通讯作者：
Wang, XD
影响因子：
9.2
作者：
Halenbeck, R;MacDonald, H;Wiiliams, LT
通讯作者：
Wiiliams, LT
影响因子：
--
作者：
Hughes A;Rando OJ
通讯作者：
Rando OJ