DNA-SEQUENCE RECOGNITION BY PAX PROTEINS - BIPARTITE STRUCTURE OF THE PAIRED DOMAIN AND ITS BINDING-SITE
DNA-SEQUENCE RECOGNITION BY PAX PROTEINS - BIPARTITE STRUCTURE OF THE PAIRED DOMAIN AND ITS BINDING-SITE
复制标题
DOI：
10.1101/gad.7.10.2048
复制
发表时间：
1993-10-01
影响因子：
10.5
通讯作者：
BUSSLINGER, M
中科院分区：
文献类型：
--
作者：
CZERNY, T;SCHAFFNER, G;BUSSLINGER, M
以前的DNA结合研究表明，一个完整的配对结构域是必需的转录因子BSAP（Pax-5）与DNA的相互作用。我们现在已经鉴定了BSAP识别序列的一个子集，其也结合到缺少配对结构域的36个羧基末端氨基酸的截短的BSAP肽。这类BSAP结合位点的序列比较使得有可能明确地对齐所有已知的BSAP结合位点，并推导出由两个不同的半位点组成的共有序列。我们在这里提出了一个模型的配对结构域的DNA相互作用，其中配对结构域是由两个子域，结合到两个半位点的DNA螺旋的同一侧相邻的大沟。通过甲基化干扰分析以及配对结构域及其识别序列的体外突变直接证明了这些半位点和两个配对结构域亚区的存在。根据它们与共有序列的匹配，两个半位点都有助于给定BSAP结合位点的总体亲和力。然而，没有一个天然存在的BSAP结合位点完全符合共有序列。相反，它们在其半位点中包含补偿性碱基变化，这解释了Pax蛋白的多功能和看似简并的DNA序列识别。BSAP和Pax-1之间的结构域交换实验表明，BSAP配对结构域的序列特异性由其氨基和羧基末端亚结构域决定。此外，仅影响两个子域之一的突变限制了配对结构域的序列特异性。此类突变之前已被证明是小鼠发育突变体（波动、斑点和小眼）和人类综合征（瓦登堡综合征和无虹膜）的原因，因此可能会通过突变的Pax蛋白差异地影响靶基因的调节。
Previous DNA-binding studies indicated that an intact paired domain is required for interaction of the transcription factor BSAP (Pax-5) with DNA. We have now identified a subset of BSAP recognition sequences that also bind to a truncated BSAP peptide lacking 36 carboxy-terminal amino acids of the paired domain. Sequence comparison of this class of BSAP-binding sites made it possible to unequivocally align all known BSAP-binding sites and to deduce a consensus sequence consisting of two distinct half sites. We propose here a model for the paired domain-DNA interaction in which the paired domain is composed of two subdomains that bind to the two half-sites in adjacent major grooves on the same side of the DNA helix. The existence of these half sites and of the two paired domain subregions was directly demonstrated by methylation interference analysis and by in vitro mutagenesis of both the paired domain and its recognition sequence. Both half-sites contribute to the overall affinity of a given BSAP-binding site according to their match with the consensus sequence. However, none of the naturally occurring BSAP-binding sites completely conform to the consensus sequence. Instead, they contain compensatory base changes in their half-sites that explain the versatile and seemingly degenerate DNA sequence recognition of Pax proteins. Domain swap experiments between BSAP and Pax-1 demonstrated that the sequence specificity of the BSAP paired domain is determined by both its amino- and carboxy-terminal subdomains. Moreover, mutations affecting only one of the two subdomains restricted the sequence specificity of the paired domain. Such mutations have been shown previously to be the cause of mouse developmental mutants (undulated, Splotch, and Small eye) and human syndromes (Waardenburg's syndrome and aniridia) and may thus differentially affect the regulation of target genes by the mutated Pax protein.