Sakaguchi Chikako, Morishita Takashi, Shinagawa Hideo, Hishida Takashi
Laboratory of Genome Dynamics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
BMC Mol Biol. 2008 Mar 3;9:27. doi: 10.1186/1471-2199-9-27.
DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage.
To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant.
These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.
DNA双链断裂（DSB）可由诸如电离辐射和化学暴露等外源性损伤诱导产生，其也可能因DNA复制叉停滞或崩溃而出现。在高等真核生物中，未能修复DSB会导致基因组不稳定、细胞死亡以及癌症。粟酒裂殖酵母fbh1基因编码一种F-box DNA解旋酶，先前研究表明该酶在Rhp51（酿酒酵母RAD51的同源物）依赖的DSB重组修复中发挥作用。DNA损伤后，与绿色荧光蛋白（GFP）融合的Fbh1定位于离散的核焦点。
为确定高度保守的F-box和解旋酶结构域的功能作用，我们对在这些结构域中携带特定突变的fbh1突变体进行了表征。我们发现F-box突变体fbh1-fb扰乱了Fbh1的核定位，赋予了类似fbh1缺失的表型。此外，即使通过与核定位信号序列融合将Fbh1-fb靶向细胞核，DNA损伤的fbh1-fb细胞中也不会形成核焦点。相比之下，解旋酶突变体fbh1-hl导致Fbh1焦点的积累，无论是否存在DNA损伤，并且赋予的损伤敏感性高于缺失等位基因。F-box的额外突变减轻了fbh1-hl突变体的超形态表型。
这些结果表明，F-box和DNA解旋酶结构域在Fbh1功能中发挥着不可或缺但又不同的作用。SCFFbh1复合物的组装对于Fbh1的核定位和DNA损伤诱导的焦点形成都是必需的，因此是Fbh1重组功能的先决条件。
