FINDING NOVEL REAGENTS BY BIOINFORMATICS AND BIOCHEMISTRY

描述（由申请人提供）：本项目旨在寻找在分子生物学研究的许多方面有用的新型限制性内切酶。一个特别的焦点将是能够识别修饰DNA的限制性内切酶，特别是含有5-甲基胞嘧啶和5-羟甲基胞嘧啶的DNA，因为这些酶在表观遗传学研究中有许多潜在的应用。识别这些修饰碱基的酶已知存在于细菌和古细菌菌株中，过去，许多酶是通过随机生化筛选发现的。在该项目中，潜在的新酶的发现将使用生物信息学技术，并利用大量目前已测序的原核生物基因组，包括完全测序的基因组和鸟枪序列。该项目将确定好的候选者，然后使用生化方法来表征它们。除了将从该筛选中发现的任何有用的酶商业化之外，所获得的有关所发现的限制性内切酶的生化功能的信息对于基因组注释将是无价的，并且将对研究致病生物的研究人员具有实际意义。后者将源于我们的努力，以表征病原体中的限制性修饰系统，并确定那些需要在转化之前通过事先修饰来敲除或绕过的系统。因此，本研究结果将对表观遗传学、发病机制和基因组注释等领域产生直接影响。

DESCRIPTION (provided by applicant): This project aims to find novel restriction enzymes that will be useful in many aspects of molecular biology research. One particular focus will be on restriction endonucleases that are able to recognize modified DNA, especially DNA containing 5-methylcytosine and 5- hydroxymethycytosine since such enzymes have many potential applications in epigenetics research. Enzymes recognizing these modified bases are known to occur in bacterial and archaea strains and in the past, many have been found by random biochemical screening. In the proposed project, the discovery of potential new enzymes will use bioinformatics techniques and take advantage of the large number of currently sequenced prokaryotic genomes, both completely sequenced genome and shotgun sequences. The project will identify good candidates and then use biochemical methods to characterize them. In addition to commercializing any useful enzymes that are found from this screen, the information gained about the biochemical function of the restriction enzymes discovered will be invaluable for genome annotation and will have practical implications for researchers working with pathogenic organisms. The latter will result from our efforts to characterize the restriction-modification systems in pathogens and identify those systems which need either to be knocked out or circumvented by prior modification prior to transformation. Thus, the results of this research will have immediate implications for the fields of epigenetics, pathogenesis and genome annotation.