IDENTIFICATION OF SMARCAL1 AS A COMPONENT OF THE DNA DAMAGE RESPONSE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 SMARCAL1（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。SMARCAL1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细胞免疫缺陷、生长抑制和肾功能障碍。然而，SMARCAL1的细胞功能尚不清楚。在这里，使用非洲爪蟾卵提取物和质谱分析，我们确定SMARCAL1作为招募双链DNA断裂的蛋白质。SMARCAL1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，SMARCAL1与RPA的物理相互作用独立于DNA。SMARCAL1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Rad51以促进同源重组修复。我们发现，从U2OS细胞SMARCAL 1的耗尽导致RAD51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SMARCAL1是一种新的DNA损伤结合蛋白，参与复制叉稳定。 本书已出版：（305） 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. 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 )