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

PAX 6是一种转录因子，通过其配对和同源DNA结合结构域与基因组结合。PAX 6的一级序列在整个进化过程中高度保守。它是眼睛发育的主要调节器。编码PAX 6的基因在多种人类眼部畸形中发生突变。人类PAX 6基因的一个拷贝的失活导致典型的无虹膜;典型的是由于视网膜、透镜和角膜的发育异常而导致的虹膜缺失和视力损害。在典型无虹膜个体的配对结构域中已经鉴定出多种不同的致病错义突变。没有明确的致病性错义变异已确定影响同源结构域。因此，PAX 6与DNA的相互作用可能是PAX 6的主要发育功能。由于我们对PAX 6在基因组中的结合位置了解有限，对PAX 6在发育过程中的功能的理解受到了限制。几个真正的PAX 6结合位点已被定义和那些已经显示出显着的多样性，在结合位点的序列，使其难以定义一个典型的motif.AimsThis项目将采用酵母单杂交（Y1 H）技术（PMID：22884952），它允许大规模并行分析不同的蛋白质的结合到一个定义的DNA序列。在MRC HGU中，Kudla实验室使用合成生物学技术构建编码任何肽中饱和氨基酸取代的质粒文库。申请人将参与创建每个残基上所有可能氨基酸取代的配对结构域文库。然后使用Y1 H检测PAX 6配对结构域的所有~2600种变体与3个经验证的脊椎动物PAX 6结合位点的结合（PMID 12710953）。这些Y1 H实验将产生一个定量测量，假设与每个变异域的DNA结合潜力相关。该项目的计算部分将试图定义Y1 H输出的结构基础。这将使用与DNA结合的PAX 6配对结构域的现有晶体结构作为骨架来模拟每个突变的四级结构的改变，并将其与配对结构域功能的实验测量相协调。对于测试的三个结合位点中的每一个，配对结构域中的四组变体将是特别感兴趣的：（超级激活剂）结合减少但可检测的那些（弱激活剂）与人类疾病相关的错义变体（致病性错义）在正常人群中发现的错义变体（良性误解）。使用计算建模和建立在最近的工作（PMID：26490019）我们还将探讨一种有趣的可能性，即PAX 6配对识别的是经典DNA螺旋形状的微妙扭曲，而不是碱基序列，或者除了碱基序列之外，我们认为，这个令人兴奋的项目将使我们能够确定在蛋白质和DNA的结构元件，介导的DNA-蛋白质相互作用的亲和力。具体而言，该项目将提高：我们对DNA配对结构域相互作用语法的理解患者致病和良性突变的区分人类基因组中功能性PAX 6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.