文献阅读笔记：同源重组修复与DNA损伤信号传导的协调

Nature Cell Biology 11, 592 - 603 (2009)
Published online: 26 April 2009 | doi:10.1038/ncb1865

RAD18 transmits DNA damage signalling to elicit homologous recombination repair

Jun Huang¹, Michael S. Y. Huen¹, Hongtae Kim^1,3, Charles Chung Yun Leung², J N Mark Glover², Xiaochun Yu⁴ & Junjie Chen¹

1.  双链断裂（DSBs）背景

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can cause cell death, mutations and chromosomal instability, which eventually lead to tumorigensis. Cells respond to DSBs by rapidly recruiting a host of proteins to chromatin regions surrounding these sites. The concentration of DNA damage and/or repair proteins at or near DSBs allows for the visualization of these proteins as ionizing radiation-induced foci (IRIF).

2. 损伤信号传递和应答的次序

Increasing evidence suggests that ATM kinase (ataxia-telangiectasia mutated)-dependent phosphorylation of the histone variant H2AX is the initial signal for subsequent accumulation of various checkpoint and repair proteins at the sites of DNA breaks7, 8. Phosphorylated H2AX binds directly to the BRCT domains of MDC1 (mediator of DNA damage checkpoint 1), which allows for RNF8 recruitment. RNF8, together with an E2 ubiquitin conjugase UBC13 promotes protein ubiquitylation at DNA damage sites, bringing about a local increase in several other mediator proteins such as BRCA1 and 53BP1, which culminates in proper checkpoint activation.

用箭头概括就是：

phosphorylation of the histone variant H2AX by ATM kinase → Phosphorylated H2AX binds directly to the BRCT domains of MDC1 →  recruitment of RNF8 → RNF8, together with UBC13 promote protein ubiquitylation at DNA damage sites → increase of several other mediator proteins such as BRCA1 and 53BP1, which culminates in proper checkpoint activation.

3. RAD51参与同源重组修复途径

DNA repair through homologous recombination requires the recombinase RAD51, and for vertebrates five RAD51 paralogues1. These RAD51 paralogues form two distinct protein complexes in vivo, a RAD51C/XRCC3 heterodimer and a RAD51B/ RAD51C/ RAD51D/XRCC2 heterotetramer. Mutation of any of these five paralogues reduces their subnuclear assembly and renders cells hypersensitive to DSBs, highlighting the importance of these RAD51 paralogues in HRR.

4. DNA修复与DNA损伤信号传导之间的cross-talk将会是研究的热点

An emerging concept in the field is that there is continuous cross-talk between DNA repair and DNA damage checkpoint pathways, however, little is known about how such coordination is achieved in the cell.

5. 文章的逻辑结构

   实验结果与数据

    ①RAD18 localizes to sites of DNA DSBs
    ②Damage-induced RAD18 foci formation requires the ubiquitin ligase RNF8 E3
    ③The zinc-finger domain of RAD18 targets it to sites of DSBs
    ④The ZNF domain of RAD18 binds directly to ubiquitin in vitro
    ⑤RAD18 promotes homologous recombination in an RNF8-dependent manner
    ⑥RAD18 interacts with RAD51C
    ⑦The ability of RAD51C to function in homologous recombination correlates with its association with RAD18
    ⑧RAD18 participates in two independent DNA damage repair pathways

  结论与讨论

    鉴于我水平有限，这里总结不出来，感兴趣的看原始文献吧。

6. 文章中用得最多的实验技术是IF。我比较感兴趣的两个是GST pull-down和TAP。IF不是不感兴趣，是暂时没条件来做，可惜。

GST pull-down assay

The GST fusion proteins were expressed in E. coli and purified as described previously. GST-fusion protein (2 μg) or GST alone was immobilized on glutathione-Sepharose 4B beads and incubated with lysates prepared from cells that were transiently transfected with plasmids encoding indicated proteins.

可以参考作者的体系，GST融合蛋白用量2 μg。

其他了解到的知识点：

⑴ RAD18 is well-known for its function in DNA damage bypass and post-replication repair in yeast and vertebrates, where it promotes monoubiquitylation of proliferating cell nuclear antigen (PCNA) at stalled replication forks.

⑵ The FHA domain of RNF8 is required for its recruitment to damage sites through its interaction with MDC1, its E3 ligase activity is required for the targeting of downstream checkpoint and repair proteins to DSBs.

⑶ The recombination protein RAD51 is the key component of the HRR machinery and the formation of Rad51 foci can be used as an indicator of HRR.

⑷ Protein ubiquitylation is emerging as an important form of covalent modification that regulates various biological processes including DNA damage signalling and repair pathways.