Czerny T, Schaffner G, Busslinger M
Research Institute of Molecular Pathology, Vienna, Austria.
Genes Dev. 1993 Oct;7(10):2048-61. doi: 10.1101/gad.7.10.2048.
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.
先前的DNA结合研究表明，转录因子BSAP（Pax-5）与DNA相互作用需要完整的配对结构域。我们现在已经鉴定出一类BSAP识别序列，它们也能与一个截短的BSAP肽结合，该肽缺少配对结构域的36个羧基末端氨基酸。对这类BSAP结合位点进行序列比较，使得明确排列所有已知的BSAP结合位点并推导由两个不同半位点组成的共有序列成为可能。我们在此提出一个配对结构域与DNA相互作用的模型，其中配对结构域由两个亚结构域组成，它们与DNA螺旋同一侧相邻大沟中的两个半位点结合。通过甲基化干扰分析以及对配对结构域及其识别序列进行体外诱变，直接证明了这些半位点和两个配对结构域亚区域的存在。根据与共有序列的匹配程度，两个半位点都对给定BSAP结合位点的整体亲和力有贡献。然而，天然存在的BSAP结合位点没有一个完全符合共有序列。相反，它们在半位点中含有补偿性碱基变化，这解释了Pax蛋白对DNA序列的广泛且看似简并的识别。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末端和羧基末端亚结构域共同决定。此外，仅影响两个亚结构域之一的突变限制了配对结构域的序列特异性。先前已证明此类突变是小鼠发育突变体（起伏、斑点和小眼）和人类综合征（瓦登伯革氏综合征和无虹膜）的病因，因此可能会不同程度地影响突变Pax蛋白对靶基因的调控。
