Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.
Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.
复制标题
激光诱导染色质和核心颗粒中组蛋白与 DNA 的交联：对研究组蛋白-DNA 相互作用的影响。
作者：
V.Yu. Stefanovsky;Stephan I. Dimitrov;V. Russanova;Dimitar Angelov;I. Pashev
UV激光照射已被用来共价交联组蛋白的DNA在核，染色质和核心颗粒和不同的组蛋白物种的存在下，在共价连接的材料进行免疫化学检测。当细胞核被照射，然后用胰蛋白酶消化以切割N-和C-末端组蛋白尾部时，没有发现组蛋白共价连接到DNA上。这一发现表明，UV激光诱导的组蛋白与DNA的交联仅通过非结构域完成。这种出乎意料的交联方式在染色质、H1耗尽的染色质和核心颗粒中起作用，即独立于染色质结构。然而，交联的效率显示出这种依赖性：虽然交联的产率在总染色质和H1耗尽的染色质中相似，但在核心颗粒中，H2 A、H2 B和H4的效率低3-4倍，H3的效率低10-12倍。降低的交联效率，特别是显着的情况下的H3，是由于减少的结合位点的数量，并分别被认为是一个直接的证据与接头DNA的核心组蛋白的非结构化尾部的相互作用。
UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.