Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state
Specificity of SAF-A and lamin B binding in vitro correlates with the satellite DNA bending state
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DOI：
10.1002/jcb.1220
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发表时间：
2001-01-01
影响因子：
4
通讯作者：
Podgornaya, OI
中科院分区：
文献类型：
--
作者：
Lobov, IB;Tsutsui, K;Podgornaya, OI
有证据表明，基质附着区（MAR）结合蛋白也结合卫星DNA（satDNA）。目前的工作的目的是确定是否主要的核基质（NM）MAR结合蛋白能够识别不同位置的satDNAs和什么样的DNA结构特征是重要的识别。在核和NM中，当使用免疫球蛋白κ基因的MAR（IG kappaMAR）和近着丝粒（periCEN）satDNA片段时，在西南印迹上识别出许多相同的多肽。然而，当人类和小鼠CEN satDNA用于探针时，结合显著降低。NM提取物经离子交换层析后，主要的DNA结合蛋白被鉴定为SAF-A（支架附着因子A）和核纤层蛋白B。不可能通过凝胶迁移率变动分析（GMSA）测试核纤层蛋白B的结合，但SAF-A显示出区分GMSA中CEN和periCEN satDNA片段的能力。虽然periCEN片段在电泳上具有异常缓慢的迁移率，这是弯曲DNA的标志，但CEN satDNA片段具有正常的迁移率。使用楔形模型（Ulanovsky和Trifonov [1987] Nature 326：720-722）进行计算机分析，该模型描述了弯曲状态如何取决于特定的核苷酸序列。该模型预测的片段的弯曲状态与它们被NM蛋白识别的能力非常一致。因此，SAF-A和核纤层蛋白B能够以与MAR结合蛋白识别MAR相同的方式识别satDNA的保守结构特征，尽管缺乏共有序列。CEN和periCEN satDNA的区别在于与这些片段的螺旋曲率相关的蛋白质。(C)2001 Wiley-Liss，Inc.
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. (C) 2001 Wiley-Liss, Inc.