Analysis of the contribution of RecQ family proteins to a maintenance system of genetic information using cell-free experiment system.

本项目的目的是通过非洲爪蟾卵提取物无细胞实验系统分析RecQ家族解旋酶及其相互作用蛋白在DNA损伤修复过程中的行为和功能。Blm基因在DNA复制过程中与染色质结合，并与DNA拓扑异构酶Ⅲ α（Top3α）相互作用。此外，在免疫耗竭Blm或Top3α后观察到DNA复制的显著抑制。进一步的分析表明，这种抑制不是由于检查点通路的激活。Werner综合征和Rothmund-Thomson综合征的致病基因产物，分别是Recql和Recql 4，在诱导DNA双链断裂（DSB）时与染色质相关。解旋酶的结合完全依赖于RPA的存在，RPA是真核细胞中的单链DNA结合蛋白复合物。为了定量分析修复DSB的活性，我构建了实验系统来测量使用线性化质粒DNA作为底物的卵提取物中的同源重组修复（HRR）和非同源末端连接（NHEJ）的活性。实验结果表明：1）NHEJ的修复效果是HRR的近1000倍; 2）NHEJ活性对aphidicolin（DNA聚合酶α、δ和ε的抑制剂）和wortmannin（DNA依赖性蛋白激酶的抑制剂）部分敏感; 3）加入aphidicolin后几乎检测不到HRR活性。作为从蛋提取物中免疫耗竭RecQL 4后的测定结果，在耗竭的提取物中观察到NHFJ活性的显著抑制和HRR活性的显著促进。因此，这表明RecQL 4通过刺激NHEJ和抑制HRR参与DSB修复。

The aim of the project was to analyze behavior and function of RecQ family helicases and their interacting proteins during repair processes of DNA damages by means of Xenopus egg extract cell free experimental system. A product of Bloom syndrome causative gene, Blm, was bound to chromatin during the process of DNA replication in the extract, and the chromatin binding of Blm was mutually dependent on chromatin loading of DNA topoisomerase III α (Top3α), which is known as one of interacting proteins of Blm. In addition, significant suppression of DNA replication was observed after an immuno-depletion of Blm or Top3α. Further analysis indicated that the suppression was not due to an activation of checkpoint pathway. Causative gene products of Werner and Rothmund-Thomson syndromes, Wrn and RecQL4, respectively, were associated onto chromatin in response to an induction of DNA double-strand breaks (DSB). The association of the helicases was completely dependent on the presence of RPA, a single-stranded DNA binding protein complex in eukaryotic cells. For quantitative analyses for activities to repair DSB, I constructed experimental systems to measure activities of homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) in the egg extract using linearized plasmid DNA as substrates. Results of the assay revealed that 1)repair through NHEJ was almost 1000-fold more effective than HRR, that 2)NHFJ activity was partially sensitive to aphidicolin, an inhibitor of DNA polymerise α, δ and ε, or wortmannin, an inhibitor of DNA-dependent protein kinase, and that 3)HRR activity was hardly detected after the addition of aphidicolin. As a result of the assay after immuno-depletion of RecQL4 from the egg extract, marked suppression of NHFJ activity and significant promotion of HRR activity were observed in the depleted extarct. Thus, it was suggested that RecQL4 is concerning to DSB repair by stimulating NHEJ and suppressing HRR.