Lobov I B, Tsutsui K, Mitchell A R, Podgornaya O I
Department of Cell Cultures, Institute of Cytology RAS, St. Petersburg 194064, Russia.
J Cell Biochem. 2001;83(2):218-29. doi: 10.1002/jcb.1220.
There is evidence that Matrix Attachment Region (MAR)-binding proteins also bind satellite DNA (satDNA). The aim of the current work was to determine whether the major nuclear matrix (NM) MAR-binding proteins are able to recognize satDNAs of different locations and what DNA structural features are important for the recognition. In nuclei and NM, a number of the same polypeptides were recognized on a southwestern blot when MAR of immunoglobulin kappa gene (Ig kappa MAR) and pericentromeric (periCEN) satDNA fragments were used. However, the binding decreased dramatically when human and mouse CEN satDNA were used for the probes. After an NM extract was subjected to ion exchange chromatography, the main DNA-binding proteins were identified as SAF-A (scaffold attachment factor A) and lamin B. It was not possible to test the binding of lamin B by gel mobility shift assay (GMSA), but SAF-A showed an ability to distinguish CEN and periCEN satDNA fragments in GMSA. While periCEN fragments have an abnormally slow mobility on electrophoresis, which is a hallmark of bent DNA, CEN satDNA fragments have a normal mobility. A computer analysis was done using the wedge model (Ulanovsky and Trifonov [1987] Nature 326:720-722), which describes how the curved state depends on particular nucleotide sequences. The curved states of the fragments predicted by the model are in good agreement with their ability to be recognized by NM proteins. Thus SAF-A and lamin B are able to recognize conserved structural features of satDNA in the same way that MAR-binding proteins recognize MARs in spite of a lack of a consensus sequence. CEN and periCEN satDNAs are distinguished by proteins in correlation with the helical curvature of these fragments.
有证据表明，基质附着区域（MAR）结合蛋白也能结合卫星DNA（satDNA）。当前研究的目的是确定主要的核基质（NM）MAR结合蛋白是否能够识别不同位置的satDNA，以及哪些DNA结构特征对于这种识别很重要。在细胞核和NM中，当使用免疫球蛋白κ基因的MAR（IgκMAR）和着丝粒周围（periCEN）satDNA片段时，在蛋白质免疫印迹法中可识别出许多相同的多肽。然而，当使用人和小鼠的着丝粒（CEN）satDNA作为探针时，结合显著减少。对NM提取物进行离子交换色谱分析后，确定主要的DNA结合蛋白为SAF-A（支架附着因子A）和核纤层蛋白B。无法通过凝胶迁移率变动分析（GMSA）检测核纤层蛋白B的结合情况，但SAF-A在GMSA中显示出区分CEN和periCEN satDNA片段的能力。虽然periCEN片段在电泳时迁移异常缓慢，这是弯曲DNA的一个标志，但CEN satDNA片段迁移正常。使用楔形模型（Ulanovsky和Trifonov [1987]《自然》326:720 - 722）进行了计算机分析，该模型描述了弯曲状态如何取决于特定的核苷酸序列。模型预测的片段弯曲状态与其被NM蛋白识别的能力高度一致。因此，尽管缺乏共有序列，SAF-A和核纤层蛋白B能够像MAR结合蛋白识别MAR一样识别satDNA的保守结构特征。CEN和periCEN satDNA通过与这些片段的螺旋曲率相关的蛋白质来区分。
