Department of Cell Biology & Plant Biochemistry, Biochemistry Centre, University of Regensburg, Universitätsstr. 31, D-93053 Regensburg, Germany.
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
J Mol Biol. 2018 Aug 17;430(17):2747-2759. doi: 10.1016/j.jmb.2018.06.046. Epub 2018 Jun 30.
Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1-2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1-2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1∆HMG completely lacking the HMG-box domain was studied. SSRP1∆HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1∆HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1-1 mutant. SSRP1∆HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.
组蛋白伴侣在真核细胞中染色质的调节结构转变中发挥关键作用，涉及核小体的解组装和重新组装。组蛋白伴侣 FACT 是一种异源二聚体复合物，由植物和后生动物中的 SSRP1/SPT16 组成，参与各种依赖于 DNA 的过程中的动态核小体重排，包括转录、复制和修复。SSRPl 亚基的 C 端 HMG 盒结构域介导体外与 DNA 和核小体的相互作用，但在体内的相关性尚不清楚。在这里，我们证明拟南芥 ssrp1-2 突变体植物表达截短的 SSRP1 蛋白。尽管截短的 HMG 盒结构域的结构明显受到干扰，但它仍然表现出残留的 DNA 结合活性，但失去了 DNA 弯曲活性。由于 ssrp1-2 植物表现出表型影响但存活，HMG 盒结构域可能在功能上是非必需的。为了检验这种可能性，研究了完全缺乏 HMG 盒结构域的 SSRP1∆HMG。体外的 SSRP1∆HMG 不能与 DNA 结合，其与核小体的相互作用也严重减少。尽管如此，该蛋白显示出与 SSRP1 相似的核迁移和蛋白相互作用。有趣的是，SSRP1∆HMG 的表达在支持非存活的拟南芥 ssrp1-1 突变体的正常生长和发育方面几乎与全长 SSRP1 一样有效。SSR1∆HMG 在结构上与真菌同源物 Pob3 相似，与 SSRP1 具有明显的相似性，但它缺乏 C 端 HMG 盒。因此，我们的研究结果表明，SSRPl 蛋白中保守的 HMG 盒结构域在拟南芥中并不关键，因此，SSRPl/SPT16 在植物/后生动物中的功能和 Pob3/Spt16 在真菌中的功能可能比预期的更相似。
