Harnessing promiscuous DNA-modification dependent enzymes used in phage defence

背景资料：噬菌体的数量是细菌的十倍，选择压力导致了噬菌体抗性系统的进化，保护细菌免受噬菌体捕食。这些系统中的许多已经被证明对生物化学家来说是无价的：限制修饰和CRISPR-cas支撑着重组DNA和基因组编辑革命。本计画将探讨噬菌体与细菌之间的持续攻击与反击。 我们最近表征了BrxU，一种高度混杂的限制性内切酶，它识别多种不同的DNA修饰，并使用任何核苷酸和多种金属来切割修饰的DNA。如何使用这些配体的细节，以及修饰识别如何导致切割，仍有待发现。此外，研究人员已经进化出BrxU家族的蛋白质抑制剂，探索这些相互作用将有助于进一步了解BrxU机制。除了这种基本的生物化学，BrxU酶具有生物技术潜力，因为它们能够识别人类基因组中高达4%的胞嘧啶DNA修饰。这些表观遗传标记在发育过程、干细胞的多能性、神经退行性疾病和肿瘤发生中具有作用。因此，BrxU可以与下一代测序（NGS）组合用于绘制DNA修饰位点。这将提供一个平台，以更好地了解表观遗传标记在发育和疾病过程中的作用。 目的：这项学术-工业联合提案将使用分子生物学、微生物学、基因组学、生物化学和结构生物学来研究BrxU。学生将（1）检查BrxU生物化学，即（i）序列特异性，（ii）DNA修饰特异性和（iii）噬菌体蛋白IPI对BrxU的抑制;（2）进行BrxU结构研究，以了解IPI对DNA修饰的识别和抑制;（3）应用BrxU通过NGS绘制DNA修饰图。

Background: Bacteriophages (phages) outnumber bacteria by ten to one, and the selection pressure has led to the evolution of phage-resistance systems that protect bacteria from phage predation. Many of these systems have proved invaluable to biochemists: restriction-modification and CRISPR-cas underpin the recombinant DNA and genome editing revolutions. This project will investigate the continuing attack and counter-attack between phage and bacteria. We have recently characterised BrxU, a highly promiscuous restriction enzyme that recognises multiple different DNA modifications, and uses any nucleotide together with a wide selection of metals, to cleave modified DNA. The details of how these ligands are used, and how modification recognition leads to cleavage, remain to be uncovered. Furthermore, phages have evolved protein inhibitors of the BrxU family and exploring these interactions will allow further understanding of the BrxU mechanism. Beyond this fundamental biochemistry, the BrxU enzymes have biotechnological potential due to their ability to recognise cytosine DNA modifications that are present in up to 4% of the human genome. These epigenetic markers have roles in developmental processes, pluripotency of stem cells, neurodegenerative diseases and tumourigenesis. BrxU could therefore be used to map DNA modification sites in combination with next generation sequencing (NGS). This will provide a platform to better understand the role of epigenetic markers in developmental and disease processes. Aims: This joint academic-industrial proposal will use molecular biology, microbiology, genomics, biochemistry and structural biology to investigate BrxU. The student will (1) examine BrxU biochemistry, namely (i) sequence specificity, (ii) DNA-modification specificity and (iii) inhibition of BrxU by phage protein IPI; (2) perform BrxU structural studies to understand DNA-modification recognition and inhibition by IPI; (3) apply BrxU to map DNA modifications via NGS.