Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A.
Unexpected DNA loss mediated by the DNA binding activity of ribonuclease A.
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核糖核酸酶的DNA结合活性介导的意外DNA丢失。
DOI：
10.1371/journal.pone.0115008
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Houseley J
中科院分区：
文献类型：
--
作者：
Donà F;Houseley J
核糖核酸酶A（RNase A）广泛用于分子生物学研究中，用于分析测定和核酸制备。RNase A的催化机制已被充分理解，并且绝对排除了对DNA的活性;然而，关于RNase A降解DNA的轶事报道并不罕见。在这里，我们描述了一种机制，其中RNase A处理可以导致明显的DNA降解。这是由于令人惊讶的发现，即RNA酶A在苯酚：氯仿混合物中仍然具有功能，据我们所知，RNA酶A是唯一能在这种高度变性的溶剂环境中存活的酶。虽然RNA酶A不切割DNA骨架，但它能够结合DNA，形成稳定的RNA酶A-DNA复合物，该复合物在苯酚：氯仿纯化期间分配到界面或有机相。RNase A DNA结合活性在苯酚中的意外存活意味着这些复合物不溶解，并且大量的RNase A结合的DNA从水相中永久除去并在相分离时损失。这种效应将影响多个程序的DNA回收，并可能代表全基因组研究中序列偏倚的来源。我们的研究结果还表明，使用RNase A进行的分析研究的结果必须小心考虑。
Ribonuclease A (RNase A) is widely used in molecular biology research both for analytical assays and for nucleic acid preparation. The catalytic mechanism of RNase A is well understood and absolutely precludes activity on DNA; however anecdotal reports of DNA degradation by RNase A are not uncommon. Here we describe a mechanism by which RNase A treatment can lead to apparent DNA degradation. This results from the surprising finding that RNase A remains functional in a phenol:chloroform mixture, to our knowledge the only enzyme that survives this highly denaturing solvent environment. Although RNase A does not cleave the DNA backbone it is capable of binding to DNA, forming stable RNase A-DNA complexes that partition to the interphase or organic phase during phenol:chloroform purification. The unexpected survival of the RNase A DNA-binding activity in phenol means that these complexes are not dissolved and a substantial amount of RNase A-bound DNA is permanently removed from the aqueous phase and lost on phase separation. This effect will impact DNA recovery from multiple procedures and is likely to represent a source of sequence bias in genome-wide studies. Our results also indicate that the results of analytical studies performed using RNase A must be considered with care.
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DOI：
10.1085/jgp.24.1.15
发表时间：
1940-01-01
期刊：
JOUR GEN PHYSIOL
影响因子：
--
作者：
KUNITZ, M.
通讯作者：
KUNITZ, M.
影响因子：
64.8
作者：
FINDLAY, D;HERRIES, DG;MATHIAS, AP
通讯作者：
MATHIAS, AP
影响因子：
--
作者：
Jones, W
通讯作者：
Jones, W
影响因子：
11.4
作者：
Martens, JHA;O'Sullivan, RJ;Jenuwein, T
通讯作者：
Jenuwein, T
影响因子：
9.2
作者：
Lehnertz, B;Ueda, Y;Peters, AHFM
通讯作者：
Peters, AHFM