Kumata Yuji, Tada Shusuke, Yamanada Yumie, Tsuyama Takashi, Kobayashi Takayuki, Dong Yu-Peng, Ikegami Kyoko, Murofushi Hiromu, Seki Masayuki, Enomoto Takemi
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
Biochim Biophys Acta. 2007 Apr;1773(4):556-64. doi: 10.1016/j.bbamcr.2007.01.005. Epub 2007 Jan 18.
Mutations in RecQL4 are a causative factor in Rothmund-Thomson syndrome, a human autosomal recessive disorder characterized by premature aging. To study the role of RecQL4, we employed a cell-free experimental system consisting of Xenopus egg extracts. RecQL4 loading onto chromatin was observed regardless of the presence or absence of EcoRI. However, in the absence of EcoRI, RecQL4 loading was suppressed by geminin, an inhibitor of pre-replicative complex formation, while in the presence of EcoRI, it was not affected. These results suggest that under the former condition, RecQL4-loading depended on DNA replication, while under the latter, the interaction occurred in response to double-stranded DNA breaks (DSBs) induced by EcoRI. DSB-induced RecQL4 loading depended on the function of the ataxia-telangiectasia mutated protein, DNA-dependent protein kinase (DNA-PK), and replication protein A, while there were only minor changes in DNA replication-associated RecQL4 loading upon suppression of these proteins. Furthermore, analyses using a chromatin-immunoprecipitation assay and quantification of gammaH2AX after induction of DSBs suggested that RecQL4 is loaded adjacent to Ku heterodimer-binding sites on damaged chromatin, and functions in the repair of DSBs.
RecQL4基因的突变是罗思蒙德-汤姆森综合征的致病因素，这是一种以早衰为特征的人类常染色体隐性疾病。为了研究RecQL4的作用，我们采用了一种由非洲爪蟾卵提取物组成的无细胞实验系统。无论有无EcoRI，均可观察到RecQL4加载到染色质上。然而，在没有EcoRI的情况下，RecQL4的加载受到geminin（一种复制前复合体形成抑制剂）的抑制，而在有EcoRI的情况下，它不受影响。这些结果表明，在前一种情况下，RecQL4的加载依赖于DNA复制，而在后一种情况下，这种相互作用是对EcoRI诱导的双链DNA断裂（DSB）作出的反应。DSB诱导的RecQL4加载依赖于共济失调毛细血管扩张突变蛋白、DNA依赖性蛋白激酶（DNA-PK）和复制蛋白A的功能，而在抑制这些蛋白后，与DNA复制相关的RecQL4加载只有微小变化。此外，使用染色质免疫沉淀分析和DSB诱导后γH2AX定量分析表明，RecQL4加载在受损染色质上Ku异二聚体结合位点附近，并在DSB修复中发挥作用。
