Thomashevski Andrei, High Anthony A, Drozd Mary, Shabanowitz Jeffrey, Hunt Donald F, Grant Patrick A, Kupfer Gary M
Department of Microbiology, University of Virginia Health System, University of Virginia, Charlottesville, Virginia 22908, USA.
J Biol Chem. 2004 Jun 18;279(25):26201-9. doi: 10.1074/jbc.M400091200. Epub 2004 Apr 13.
Fanconi anemia (FA) is an autosomal recessive disease marked by congenital defects, bone marrow failure, and cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA crosslinking agents such as mitomycin C. The molecular mechanism for the disease remains elusive, but at least 6 FA proteins are known to be part of what is termed the FA core complex. We used affinity pulldown of FLAG-FANCA to pull down the FA complex from whole-cell extracts. Mass spectroscopy detected previously reported FA-binding proteins, including FANCA, FANCC, FANCG, cdc2, and GRP94, thus validating the approach. We further describe a method of purification of the FA core complex in an effort to find novel complex components and biochemical activity to define the function of the complex. By using conventional chromatographic fractionation of subcellular preparations, we report: (i) the FA core complex exists in a cytoplasmic form at 500-600 kDa; (ii) a larger, 750-kDa cytoplasmic form is seen only at mitosis; (iii) a nuclear form achieves a size of 2 megaDaltons; and (iv) a distinct 1-megaDalton FA core complex exists bound to chromatin that contains phosphorylated FANCA after undergoing DNA damage. We are continuing our analysis using mass spectroscopy in an effort to characterize novel binding proteins. These data will help define the biochemical role of the FA core complex in normal cell physiology as well as in the development of the FA disease state.
范可尼贫血(FA)是一种常染色体隐性疾病,其特征为先天性缺陷、骨髓衰竭和癌症易感性。FA细胞对诸如丝裂霉素C等DNA交联剂表现出典型的超敏反应。该疾病的分子机制仍不清楚,但已知至少6种FA蛋白是所谓FA核心复合物的一部分。我们利用FLAG-FANCA的亲和下拉法从全细胞提取物中下拉FA复合物。质谱检测到先前报道的FA结合蛋白,包括FANCA、FANCC、FANCG、cdc2和GRP94,从而验证了该方法。我们进一步描述了一种纯化FA核心复合物的方法,以寻找新的复合物成分和生化活性来定义该复合物的功能。通过对亚细胞制剂进行常规色谱分离,我们报告:(i)FA核心复合物以500 - 600 kDa的细胞质形式存在;(ii)仅在有丝分裂时可见一种更大的、750 kDa的细胞质形式;(iii)一种核形式达到2兆道尔顿的大小;(iv)在DNA损伤后,一种独特的1兆道尔顿的FA核心复合物与含有磷酸化FANCA的染色质结合存在。我们正在继续使用质谱进行分析,以鉴定新的结合蛋白。这些数据将有助于确定FA核心复合物在正常细胞生理学以及FA疾病状态发展中的生化作用。