IDENTIFICATION OF SMARCAL1 AS A COMPONENT OF THE DNA DAMAGE RESPONSE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 SMARCAL 1（也称为HARP）是一种SWI/SNF家族蛋白，具有由含有单链和双链区域的DNA刺激的ATP酶活性。SMARCAL 1突变与Schimke免疫性骨发育不良相关，这是一种多系统常染色体隐性遗传疾病，其特征为T细胞免疫功能障碍、生长抑制和肾功能障碍。然而，SMARCAL 1的细胞功能尚不清楚。在这里，使用爪蟾卵提取物和质谱分析，我们确定SMARCAL 1作为招募双链DNA断裂的蛋白质。SMARCAL 1与卵提取物和人类细胞中的双链断裂和停滞的复制叉结合，特别是与单链DNA结合因子RPA共定位。此外，SMARCAL 1与RPA的物理相互作用独立于DNA。SMARCAL 1以咖啡因敏感的方式磷酸化，以响应双链断裂和停滞的复制叉。有人认为，停滞的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Rad 51以促进同源重组修复。我们发现，从U2 OS细胞SMARCAL 1的耗尽导致RAD 51焦点的频率增加后，停滞的复制叉的产生，表明叉故障是更普遍的SMARCAL 1的情况下。我们提出SMARCAL 1是一种新的DNA损伤结合蛋白，参与复制叉稳定。 本作品已出版：（L。Postow，E.M.喔，B.T. Chait，H. Funabiki“Identification of SMARCAL 1 as a component of the DNA damage response”J Biol Chem.284（2009）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 )