Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences
Bloom DNA Helicase Facilitates Homologous Recombination between Diverged Homologous Sequences
复制标题
DOI：
10.1074/jbc.m109.029348
复制
发表时间：
2009-09-25
影响因子：
4.8
通讯作者：
Hirota, Kouji
中科院分区：
文献类型：
--
作者：
Kikuchi, Koji;Abdel-Aziz, H. Ismail;Hirota, Kouji
由 Bloom DNA 解旋酶 (Blm) 失活引起的 Bloom 综合征的特征是姐妹染色单体交换、与交叉相关的同源重组 (HR) 水平增加。因此，人们相信 Blm 起到抗重组酶的作用。同时，在果蝇中，DmBlm 是促进合成依赖性链退火 (SDSA) 所必需的，这是一种与交换无关的 HR 类型。然而，高等真核生物对 SDSA 中 Blm 功能的保护一直存在争议。在这里，我们证明了 Blm 在鸡 DT40 B 淋巴细胞系 SDSA 型 HR 中的功能，其中 Ig 基因转换通过不同同源片段之间的基因内 HR 使免疫球蛋白 V 基因多样化。该反应是由激活诱导的胞苷脱氨酶介导的 V 基因尿嘧啶形成引发的，V 基因又转化为脱碱基位点，可能导致单链缺口。 BLM-/- 细胞中 Ig 基因转换频率显着降低。此外，BLM-/-细胞使用有限的供体片段，与Ig基因转换事件中的其他片段相比具有更高的同一性，这表明Blm可以促进不同序列之间的HR。为了进一步了解 Blm 在不同同源序列之间 HR 中的作用，我们测量了由 I-SceI 核酸内切酶介导的双链断裂诱导的基因靶向频率。随着双链断裂位点异源序列数量的增加，BLM-/- 细胞在基因靶向频率方面表现出更严重的缺陷。相反，Blm 的过度表达，即使是 ATP 酶缺陷的突变体，也会强烈刺激基因靶向。总之，Blm 通过一种新的不依赖于 ATP 酶的机制促进不同序列之间的 HR。
Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. Here, we demonstrate the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM-/- cells. In addition, BLM-/- cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, we measured the frequency of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM-/- cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism.