IDENTIFICATION OF SMARCAL1 AS A COMPONENT OF THE DNA DAMAGE RESPONSE

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 SMARCAL1(又称HARP)是一种受DNA单链和双链DNA刺激的具有ATPase活性的SWI/SNF家族蛋白。SMARCAL1的突变与Schimke免疫性骨发育不良症有关，Schimke免疫性骨发育不良是一种以T细胞免疫功能障碍、生长抑制和肾功能障碍为特征的多系统常染色体隐性遗传病。然而，SMARCAL1的细胞功能尚不清楚。在这里，使用非洲爪哇卵提取液和质谱仪，我们鉴定SMARCAL1是一种招募到双链DNA断裂的蛋白质。SMARCAL1结合在鸡蛋提取物和人类细胞中的双链断裂和停滞的复制叉子上，特别是与单链DNA结合因子RPA共定位。此外，SMARCAL1独立于DNA与RPA进行物理交互。SMARCAL1以咖啡因敏感的方式被磷酸化，以响应双链断裂和停滞的复制叉子。已经有人提出，停滞的分叉可以通过涉及咖啡因敏感激酶的机制来稳定，或者它们崩溃并随后招募RAD51来促进同源重组修复。我们表明，U2OS细胞中SMARCAL1的耗尽导致RAD51焦点的频率增加，从而产生停滞的复制叉子，这表明在没有SMARCAL1的情况下，叉子崩溃更普遍。我们认为SMARCAL1是一个新的DNA损伤结合蛋白，参与复制分叉的稳定。 这项工作已经发表：(L.Postow，E.M.Woo，B.T.Chait，H.Funabiki：作为DNA损伤响应的一个组件的SMARCAL1的鉴定》J Biol Chem。(2009年)35951-61)

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immune disfunction, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization. This work has been published: (L. Postow, E.M. Woo, B.T. Chait, H. Funabiki "Identification of SMARCAL1 as a component of the DNA damage response" J Biol Chem. 284(2009) 35951-61 )