Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Biomolecules. 2021 Nov 19;11(11):1724. doi: 10.3390/biom11111724.
histone-like protein HU has been shown to interact with different topological forms of DNA. Using radiolabeled HU, we examine the effects of DNA supercoiling on HU-DNA interactions. We show that HU binds preferentially to negatively supercoiled DNA and that the affinity of HU for DNA increases with increases in the negative superhelical density of DNA. Binding of HU to DNA is most sensitively influenced by DNA supercoiling within a narrow but physiologically relevant range of superhelicity ( = -0.06-0). Under stoichiometric binding conditions, the affinity of HU for negatively supercoiled DNA ( = -0.06) is more than 10 times higher than that for relaxed DNA at physiologically relevant HU/DNA mass ratios (e.g., 1:10). This binding preference, however, becomes negligible at HU/DNA mass ratios higher than 1:2. At saturation, HU binds both negatively supercoiled and relaxed DNA with similar stoichiometries, i.e., 5-6 base pairs per HU dimer. In our chemical crosslinking studies, we demonstrate that HU molecules bound to negatively supercoiled DNA are more readily crosslinked than those bound to linear DNA. At in vivo HU/DNA ratios, HU appears to exist predominantly in a tetrameric form on negatively supercoiled DNA and in a dimeric form on linear DNA. Using a DNA ligase-mediated nick closure assay, we show that approximately 20 HU dimers are required to constrain one negative supercoil on relaxed DNA. Although fewer HU dimers may be needed to constrain one negative supercoil on negatively supercoiled DNA, our results and estimates of the cellular level of HU argue against a major role for HU in constraining supercoils in vivo. We discuss our data within the context of the dynamic distribution of the HU protein in cells, where temporal and local changes of DNA supercoiling are known to take place.
组蛋白样蛋白 HU 已被证明与不同拓扑形式的 DNA 相互作用。使用放射性标记的 HU，我们研究了 DNA 超螺旋对 HU-DNA 相互作用的影响。我们表明 HU 优先结合负超螺旋 DNA，并且 HU 与 DNA 的亲和力随 DNA 负超螺旋密度的增加而增加。HU 与 DNA 的结合对 DNA 超螺旋的影响最为敏感，其范围狭窄但在生理相关的超螺旋范围内（= -0.06-0）。在化学计量结合条件下，HU 与负超螺旋 DNA 的亲和力（= -0.06）比生理相关 HU/DNA 质量比（例如 1:10）下松弛 DNA 的亲和力高 10 倍以上。然而，这种结合偏好在 HU/DNA 质量比高于 1:2 时变得可以忽略不计。在饱和时，HU 以相似的化学计量比结合负超螺旋和松弛 DNA，即每个 HU 二聚体结合 5-6 个碱基对。在我们的化学交联研究中，我们证明结合到负超螺旋 DNA 的 HU 分子比结合到线性 DNA 的 HU 分子更容易交联。在体内 HU/DNA 比下，HU 似乎主要以四聚体形式存在于负超螺旋 DNA 上，以二聚体形式存在于线性 DNA 上。使用 DNA 连接酶介导的缺口封闭测定法，我们表明大约需要 20 个 HU 二聚体来限制松弛 DNA 上的一个负超螺旋。尽管在负超螺旋 DNA 上限制一个负超螺旋可能需要更少的 HU 二聚体，但我们的结果和对细胞内 HU 水平的估计表明，HU 在体内限制超螺旋的作用不大。我们在细胞中 HU 蛋白的动态分布的背景下讨论我们的数据，已知 DNA 超螺旋的时间和局部变化会发生在这种情况下。
