Almarza Oscar, Núñez Daniel, Toledo Hector
Department of Molecular and Cellular Biology, Laboratory of Molecular Microbiology, ICBM, Faculty of Medicine, University of Chile, Avda. Independencia, 1027, Santiago, Chile.
Helicobacter. 2015 Feb;20(1):29-40. doi: 10.1111/hel.12171. Epub 2014 Sep 25.
Bacterial genomes are compacted by association with histone-like proteins to form a complex known as bacterial chromatin. The histone-like protein HU is capable of binding and bending the DNA molecule, a function related to compaction, protection, and regulation of gene expression. In Helicobacter pylori, HU is the only histone-like protein described so far. Proteomic analysis from our laboratory showed that this protein is overexpressed under acidic stress.
We used a purified recombinant wild-type protein and two mutant proteins with the amino acid substitutions K3A/S27D and K62R/V63N/P64A to characterize the function of the N-terminal domain and the flexible arm of HU.
In vitro assays for DNA protection, bending, and compaction were performed. We also designed a H. pylori hup::cat mutant strain to study the role of HU in the acid stress response. HUwt protein binds DNA and promotes its bending and compaction. Compared with the wild-type protein, both mutant proteins have less affinity for DNA and an impaired bending and compaction ability. By using qRT-PCR, we confirmed overexpression of two genes related to acid stress response (ureA and speA). Such overexpression was abolished in the hup::cat strain, which shows an acid-sensitive phenotype.
Altogether, we have shown that HUwt -DNA complex formation is favored under acidic pH and that the complex protects DNA from endonucleolytic cleavage and oxidative stress damage. We also showed that the amino-terminal domain of HU is relevant to DNA-protein complex formation and that the flexible arm of HU is involved in the bending and compaction activities of HU.
细菌基因组通过与类组蛋白结合而压缩，形成一种称为细菌染色质的复合物。类组蛋白HU能够结合并弯曲DNA分子，该功能与基因表达的压缩、保护和调控有关。在幽门螺杆菌中，HU是迄今为止所描述的唯一类组蛋白。我们实验室的蛋白质组学分析表明，这种蛋白质在酸性应激下会过度表达。
我们使用纯化的重组野生型蛋白以及两种具有氨基酸替代K3A/S27D和K62R/V63N/P64A的突变蛋白来表征HU的N端结构域和柔性臂的功能。
进行了DNA保护、弯曲和压缩的体外测定。我们还设计了一株幽门螺杆菌hup::cat突变株，以研究HU在酸应激反应中的作用。HUwt蛋白结合DNA并促进其弯曲和压缩。与野生型蛋白相比，两种突变蛋白对DNA的亲和力较低，弯曲和压缩能力受损。通过定量逆转录聚合酶链反应（qRT-PCR），我们证实了两个与酸应激反应相关的基因（ureA和speA）的过度表达。在显示酸敏感表型的hup::cat菌株中，这种过度表达被消除。
总之，我们已经表明，在酸性pH下有利于形成HUwt-DNA复合物，并且该复合物可保护DNA免受核酸内切酶切割和氧化应激损伤。我们还表明，HU的氨基末端结构域与DNA-蛋白质复合物的形成有关，并且HU的柔性臂参与了HU的弯曲和压缩活性。
