BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination.
BRCA1 and CtIP Are Both Required to Recruit Dna2 at Double-Strand Breaks in Homologous Recombination.
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DOI：
10.1371/journal.pone.0124495
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Sasanuma H
中科院分区：
文献类型：
--
作者：
Hoa NN;Kobayashi J;Omura M;Hirakawa M;Yang SH;Komatsu K;Paull TT;Takeda S;Sasanuma H
同源重组在双链断裂(DSB)的修复中起着关键作用，从而显著提高细胞对放射治疗和某些化疗的耐受性。通过同源重组的DSB修复是由5‘到3’链切除(DSB切除)启动的，核酸酶在DSB位点产生3‘单链DNA(3’ssDNA)。酿酒酵母的遗传学研究表明，DSB切除分两步进行，其中CtIP和Mre11核酸酶进行短期DSB切除，然后由DNA2和Exo1核酸酶进行远程DSB切除。最近的研究表明，CtIP通过其非催化作用而不是作为核酸酶对DSB的清除起作用。然而，目前尚不清楚CtIP对DSB切除的作用。为了探索DNA2的非催化作用，我们建立了一种免疫细胞化学方法来检测电离辐射(IR)诱导的DNA2在鸡DT40和人细胞系DSB处的亚核焦点形成，从而研究了DNA2的动力学。电离辐射仅在野生型细胞中诱导出DNA2灶，而在DNA2耗竭细胞中未见，在IR后30min达到最大值，120min时几乎检测不到。在G2期细胞中可检测到诱导灶，但在G1期细胞中未检测到诱导灶。这些观察表明，DNA2病灶代表了DNA2向DSB部位的募集以进行DSB切除。重要的是，CtIP的缺失抑制了DNA2在人细胞和鸡DT40细胞中的DSB位点的募集。同样，乳腺癌1(BRCA1)中的一个缺陷，它与CtIP物理上相互作用，并有助于DSB切除，也抑制了DNA2的招募。此外，CtIP与DNA2发生物理结合，这种结合被IR增强。我们得出结论，BRCA1和CtIP通过将DNA2招募到损伤部位来促进DSB切除，从而确保有效的同源重组所必需的DSB切除。
Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.
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影响因子：
4.5
作者：
Qing Y;Yamazoe M;Hirota K;Dejsuphong D;Sakai W;Yamamoto KN;Bishop DK;Wu X;Takeda S
通讯作者：
Takeda S
影响因子：
16
作者：
Peterson SE;Li Y;Wu-Baer F;Chait BT;Baer R;Yan H;Gottesman ME;Gautier J
通讯作者：
Gautier J
影响因子：
9.2
作者：
Paull, TT;Rogakou, EP;Bonner, WM
通讯作者：
Bonner, WM
影响因子：
10.5
作者：
Nimonkar, Amitabh V.;Genschel, Jochen;Kowalczykowski, Stephen C.
通讯作者：
Kowalczykowski, Stephen C.
由 53BP1-RIF1 和 BRCA1-CtIP 组成的细胞周期依赖性调节回路控制 DNA 修复途径的选择。
影响因子：
16
作者：
Escribano-Diaz, Cristina;Orthwein, Alexandre;Durocher, Daniel
通讯作者：
Durocher, Daniel