IDENTIFICATION OF SMARCAL1 AS A COMPONENT OF THE DNA DAMAGE RESPONSE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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细胞免疫缺陷、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。 这部著作已出版：(305。吴永明，B.T.Chait，H.Funabiki，<SMARCAL1作为DNA损伤反应的一种成分的鉴定>生物化学杂志.(2009年)35951-61)

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 immunodeficiency, 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 ha been published: (305. 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 )