Linker histones (LHs) bind to nucleosomes with their globular domain (gH) positioned in either an on- or an off-dyad binding mode. Here, we study the effect of the linker DNA (L-DNA) sequence on the binding of a full-length LH, Xenopus laevis H1.0b, to a Widom 601 nucleosome core particle (NCP) flanked by two 40 bp long L-DNA arms, by single-pair FRET spectroscopy. We varied the sequence of the 11 bp of L-DNA adjoining the NCP on either side, making the sequence either A-tract, purely GC, or mixed with 64% AT. The labeled gH consistently exhibited higher FRET efficiency with the labeled L-DNA containing the A-tract than that with the pure-GC stretch, even when the stretches were swapped. However, it did not exhibit higher FRET efficiency with the L-DNA containing 64% AT-rich mixed DNA when compared to the pure-GC stretch. We explain our observations with a model that shows that the gH binds on dyad and that two arginines mediate recognition of the A-tract via its characteristically narrow minor groove. To investigate whether this on-dyad minor groove-based recognition was distinct from previously identified off-dyad major groove-based recognition, a nucleosome was designed with A-tracts on both the L-DNA arms. One A-tract was complementary to thymine and the other to deoxyuridine. The major groove of the thymine-tract was lined with methyl groups that were absent from the major groove of the deoxyuridine tract. The gH exhibited similar FRET for both these A-tracts, suggesting that it does not interact with the thymine methyl groups exposed on the major groove. Our observations thus complement previous studies that suggest that different LH isoforms may employ different ways of recognizing AT-rich DNA and A-tracts. This adaptability may enable the LH to universally compact scaffold-associated regions and constitutive heterochromatin, which are rich in such sequences.

接头组蛋白 (LH) 与核小体结合，它们的球状结构域 (gH) 以开启或关闭二元结合模式定位。在这里，我们研究了接头 DNA (L-DNA) 序列对全长 LH、非洲爪蟾结合的影响H1.0b，通过单对 FRET 光谱分析，连接到两侧有两个 40 bp 长 L-DNA 臂的 Widom 601 核小体核心颗粒 (NCP)。我们改变了与 NCP 相邻的 11 bp 的 L-DNA 序列，使序列要么是 A-tract，要么是纯 GC，要么与 64% AT 混合。与纯 GC 拉伸相比，标记的 gH 与含有 A-tract 的标记 L-DNA 始终表现出更高的 FRET 效率，即使当拉伸被交换时也是如此。然而，与纯 GC 拉伸相比，含有 64% 富含 AT 的混合 DNA 的 L-DNA 并没有表现出更高的 FRET 效率。我们用一个模型解释了我们的观察结果，该模型显示 gH 结合在二元组上，并且两个精氨酸通过其特有的狭窄小沟介导对 A 束的识别。为了研究这种基于二元小沟的识别是否不同于先前确定的基于非二元大沟的识别，设计了一个核小体，在两个 L-DNA 臂上都有 A-束。一个A-束与胸腺嘧啶互补，另一个与脱氧尿苷互补。胸腺嘧啶束的大沟内衬有脱氧尿苷束大沟中不存在的甲基。对于这两个 A 束，gH 表现出相似的 FRET，表明它不与暴露在大沟上的胸腺嘧啶甲基相互作用。因此，我们的观察补充了先前的研究，这些研究表明不同的 LH 同种型可能采用不同的方式识别富含 AT 的 DNA 和 A-束。这种适应性可能使 LH 能够普遍紧凑的支架相关区域和组成型异染色质，