Does BLM helicase unwind nucleosomal DNA?
Does BLM helicase unwind nucleosomal DNA?
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BLM 解旋酶会解旋核小体 DNA 吗？
DOI：
10.1139/o09-051
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Zlatanova,Jordanka
中科院分区：
文献类型：
--
作者：
Fujimoto,Satoru;Tomschik,Miroslav;Zlatanova,Jordanka
RecQ解旋酶通过解析几种高度特异性的DNA结构来维持染色体稳定性。BLM是Bloom综合征中突变的蛋白质，是RecQ解旋酶家族的成员，具有DNA解旋和链退火活性。在这项研究中，我们研究了解旋活性的BLM对核小体DNA，天然核底物的酶。我们产生了一个DNA模板，包括一个强大的核小体定位序列两侧的叉状DNA，据报道，这是一个优选的BLM的DNA底物。BLM不具有可检测的解旋活动对分叉的DNA基板。然而，截短的BLM，缺乏退火活性，解开它部分。在单链DNA结合蛋白RPA的存在下，全长和截短的BLM的解旋活性得到促进。接下来，将组蛋白八聚体重构到叉状DNA上以产生叉状单体。全长BLM没有解开核小体DNA，但截短BLM解开它部分。RPA没有显著促进单体的解旋活性。这些结果表明，全长BLM可能需要额外的因素，以解开核小体DNA在体内，RPA是，就其本身而言，无法执行这一辅助功能。
RecQ helicases maintain chromosome stability by resolving several highly specific DNA structures. BLM, the protein mutated in Bloom’s syndrome, is a member of the RecQ helicase family, and possesses both DNA-unwinding and strand-annealing activity. In this study, we have investigated the unwinding activity of BLM on nucleosomal DNA, the natural nuclear substrate for the enzyme. We generated a DNA template including a strong nucleosome-positioning sequence flanked by forked DNA, which is reportedly one of the preferred DNA substrates for BLM. BLM did not possess detectable unwinding activity toward the forked DNA substrate. However, the truncated BLM, lacking annealing activity, unwound it partially. In the presence of the single-stranded DNA-binding protein RPA, the unwinding activity of both the full-length and the truncated BLMs was promoted. Next, the histone octamer was reconstituted onto the forked DNA to generate a forked mononucleosome. Full-length BLM did not unwind the nucleosomal DNA, but truncated BLM unwound it partially. The unwinding activity for the mononucleosome was not promoted dramatically with RPA. These results indicate that full-length BLM may require additional factors to unwind nucleosomal DNA in vivo, and that RPA is, on its own, unable to perform this auxiliary function.