Kinebuchi Takashi, Kagawa Wataru, Kurumizaka Hitoshi, Yokoyama Shigeyuki
Protein Research Group, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.
J Biol Chem. 2005 Aug 5;280(31):28382-7. doi: 10.1074/jbc.M503372200. Epub 2005 May 25.
The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.
DMC1蛋白是RecA的真核同源物，与RAD51具有显著的氨基酸同源性，它呈现出两种寡聚DNA结合形式，即八聚体环和螺旋丝。在八聚体环形式的晶体结构中，DMC1的N端结构域（1 - 81个氨基酸残基）具有高度灵活性，存在多种构象。另一方面，Rad51的N端结构域在螺旋丝结构中与相邻的ATP酶结构域发生特异性相互作用。为了深入了解DMC1 N端结构域的功能作用，我们制备了一个缺失突变体DMC1-(82 - 340)，它缺失了人DMC1蛋白的N端81个氨基酸残基。分析超速离心实验表明，全长DMC1形成八聚体，而DMC1-(82 - 340)是七聚体。此外，DNA结合实验表明，DMC1-(82 - 340)在单链和双链DNA结合活性方面均完全缺陷。因此，DMC1的N端结构域是形成八聚体所必需的，这可能支持DMC1蛋白适当的DNA结合活性。
