Quantitative approaches to DNA-protein interactions using synthetic biology, high-throughout yeast one-hybrid assays and structural modelling of prote

Pax6是一种转录因子，通过其配对的同源DNA结合域与基因组结合。PAX6的初级序列在整个进化过程中高度保守。它作为眼睛发育的主要调节器发挥作用。编码PAX6的基因在多种人眼畸形中发生突变。在人类中，PAX6基因的一个拷贝失活会导致经典的无虹膜；典型的是由于视网膜、晶状体和角膜的发育异常而导致的虹膜缺失和视力障碍。在典型无虹膜患者的配对区域中，已经发现了多种不同的致病错义突变。目前还没有确定影响同源域的致病错义变异体。因此，PAX6的大部分发育功能很可能是由配对结构域与DNA的相互作用造成的，我们对PAX6在发育过程中如何发挥作用的理解受到了我们对它与基因组中哪里结合的有限了解的阻碍。本项目将采用酵母单杂交(Y1H)技术(pID：22884952)，该技术允许大规模并行分析不同蛋白质与已定义的dna序列的结合。在MRC HGU内，库德拉实验室使用合成生物学技术来构建编码任何多肽中饱和氨基酸替代的质粒库。申请者将参与创建一个配对的结构域文库，其中包含每个残基上所有可能的氨基酸替换。然后将使用Y1H来测试PAX6配对结构域的所有~2600个变体与三个已验证的脊椎动物PAX6结合位点(PMID 12710953)的结合。这些Y1H实验将产生一个定量测量，假设与每个变异结构域的DNA结合潜力相关。这个项目的计算部分将试图定义Y1H输出的结构基础。这将使用与DNA结合的PAX6配对结构域的现有晶体结构作为主干，为每个突变的四元结构变化建模，并将其与配对结构域功能的实验测量相一致。对于测试的三个结合位点中的每一个，在配对结构域中的四组变体将是特别感兴趣的：那些具有比野生型(超级激活子)更强的dna结合活性的那些具有减少但可检测到的结合(弱激活子)错义变体的那些与人类疾病相关的错义变体(致病错义)在正常人群中发现的错义变体(良性错义)。利用计算模型和建立在最近的工作(PMID：26490019)的基础上，我们还将探索一种有趣的可能性，即它是经典dna螺旋形状的微妙扭曲，而不是或除了由PAX6配对结构域识别的碱基序列之外。我们认为这个令人兴奋的项目将允许我们确定蛋白质和DNA中调节DNA-蛋白质相互作用亲和力的结构元素。具体地说，这个项目将改善：我们对DNA配对结构域相互作用的语法的理解，患者致病突变和良性突变的区分，以及对人类基因组中功能性PAX6结合位点的识别。

PAX6 is a transcription factor, which binds to the genome via its paired and homeo DNA binding domains. The primary sequence of PAX6 is highly conserved throughout evolution. It functions as a master regulator of eye development. The gene encoding PAX6 is mutated in a variety of human eye malformations. Inactivation of one copy of the PAX6 gene in humans result in classical aniridia; typified by absence of the iris and visual impairment as a result of a developmental anomalies of the retina, lens and cornea. Multiple different causative missense mutations have been identified in the paired domain in individuals with classical aniridia. No definitely pathogenic missense variants have been identified affecting the homeo domain. It thus seems likely that interaction of paired domain with DNA is responsible for most of the developmental function of PAX6.Our understanding of how PAX6 functions during development has been hampered by our limited knowledge of where it binds in the genome. Few bone fide PAX6 binding sites have been defined and those that have show remarkable diversity in the sequence of the binding site making it difficult to define a canonical motif.AimsThis project will employ yeast one-hybrid (Y1H) technology (PMID: 22884952), which allows massively parallel analysis of the binding of different proteins to a defined DNA sequence. Within MRC HGU the Kudla lab use synthetic biology technology to construct libraries of plasmids encoding saturated amino acid substitutions in any peptide. The applicant will be involved in the creation of a paired domain library of all possible amino acid substitutions at each residue. Y1H will then be used to test the binding of all ~2600 variants of the PAX6 paired domain to three validated vertebrate PAX6 binding sites (PMID 12710953). These Y1H experiments will generate a quantitative measurement assumed to correlate with the DNA binding potential of each variant domain.The computational component of this project will attempt to define the structural basis of the Y1H output. This will use the existing crystal structure of the PAX6 paired domain bound to DNA as a backbone to model the alterations in the quaternary structure for each mutation and reconcile this with the experimental measurement of paired domain function. For each of the three binding sites tested, four groups of variants in the paired domain will be of specific interest:Those with DNA binding activity that is stronger that wild type (super-activators)Those with reduced but detectable binding (weak activators)Missense variants associated with human disease (pathogenic missense)Missense variants found in the normal population (benign missense).Using computational modelling and building on recent work (PMID:26490019) we will also explore the intriguing possibility that it is subtle distortions of the classic DNA helical shape rather than or in addition to base sequence that is recognised by the PAX6 paired domain.We consider that this exciting project will allow us to identify structural elements in both the protein and DNA that mediate the affinity of DNA-protein interaction. Specifically, this project will improve:Our understanding of the grammar of DNA-paired domain interactionsThe differentiation of disease-causing and benign mutations in patientsThe recognition of functional PAX6 binding sites in the human genome.