Ivanchenko M, Zlatanova J, Varga-Weisz P, Hassan A, van Holde K
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-7305, USA.
Proc Natl Acad Sci U S A. 1996 Jul 9;93(14):6970-4. doi: 10.1073/pnas.93.14.6970.
The effect of histone H1 binding on the cleavage of superhelical plasmids by single-strand-specific nucleases was investigated. Mapping of P1 cleavage sites in pBR322, achieved by EcoRI digestion after the original P1 attack, showed an intriguing phenomenon: preexisting susceptible sites became "protected," whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-strand-specific nuclease, S1. Disappearance of cutting at preexisting sites and appearance of new sites was also observed in a derivative plasmid that contains a 36-bp stretch of alternating d(AT) sequence that is known to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)18-containing plasmid led to protection of all preexisting sites except the d(AT)18 inserts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explained by long-range effects of histone H1 binding on the superhelical torsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific sequences in Pl-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-terminal tails is indicated by the fact that titration with the globular domain of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.
研究了组蛋白H1结合对单链特异性核酸酶切割超螺旋质粒的影响。通过原始P1攻击后进行EcoRI消化来绘制pBR322中P1切割位点图谱，结果显示了一个有趣的现象：预先存在的敏感位点变得“受保护”，而在高水平的H1存在时出现了一些新位点。用另一种单链特异性核酸酶S1也得到了类似的结果。在一个含有36个碱基对的交替d(AT)序列的衍生质粒中也观察到了在预先存在的位点切割的消失和新位点的出现，已知该序列会采用改变的P1敏感构象。另一方面，对含有d(AT)18的质粒二聚体进行H1滴定导致除d(AT)18插入片段外的所有预先存在的位点都受到保护，即使在高H1水平下d(AT)18插入片段仍被切割；在该质粒中没有出现新位点。预先存在的位点受到保护最好用组蛋白H1结合对质粒超螺旋扭转的长程效应来解释。另一方面，新位点的出现可能还涉及由于H1直接结合到特定序列而使处于P1敏感构象的特定序列稳定的局部效应。用H5的球状结构域进行滴定虽然导致预先存在的位点消失，但不会导致任何新位点的出现，这一事实表明这种结合涉及连接组蛋白的N端和/或C端尾巴。
