Ancient biological functions of present day DNA-damaging enzymes involved in the immune system

这项研究计划的目标是了解鱼类DNA损伤过程的形式和功能。人类免疫系统使用突变和双链DNA断裂(DSB)形式的DNA损伤来启动淋巴细胞中抗原受体基因的多样化。我们的中心假设是，鱼类中这些DNA损伤酶的同源版本参与了免疫系统以外的生物角色，可能参与了表观基因组的重新编程和发育。 所有的生物都有多条DNA损伤感知、检查点和修复途径。因此，细胞将大量精力投入到对其DNA序列的忠实维护和修复上。从这个角度来看，有颌骨脊椎动物的免疫系统是一个例外。这是所有生物学中细胞在正常发育和功能过程中主动突变并以特定部位的方式物理重组自己的基因组DNA的唯一例子。免疫系统对DNA损伤的利用被认为最早出现在软骨鱼身上。 我们最近发现，与人类免疫系统有关的一些DNA损伤酶的同源版本在特定的鱼类物种中具有不同的活性图谱，从而调节完全在免疫系统之外的功能。基于这一发现，我们提出了一项新的研究计划，以广泛地研究无颌脊椎动物七鳃鳗、软骨鱼鲨鱼以及包括大西洋鳕鱼、鲑鱼和腔棘鱼在内的几种硬骨鱼的DNA损伤酶及其生物学功能。我们的目标是1)克隆和鉴定这些物种中的DNA损伤酶，2)检测这些酶在免疫刺激和发育过程中的组织特异性表达模式，3)表达、纯化和检测鱼类DNA损伤酶之间的生化差异，以及4)利用转基因细胞和转基因动物模型比较鱼类DNA损伤酶的生物学功能。 如果我们的假设是正确的，我们预计会发现，与哺乳动物或彼此相比，这些鱼类中的一些或所有DNA损伤酶具有不同的组织表达模式和/或不同的生化特性。我们的假设还预测，当这些酶在细胞系或转基因动物中表达时，可以促使不同的DNA损伤反应，并执行不同的生物学功能。 这一研究项目代表了我的实验室的一个新方向，我们已经启动了与鱼类生物学和免疫学专家的地方、国家和国际合作。无论结果是否支持我们的假设，我们都将对鱼类DNA损伤酶的形式和功能有一个全面的了解。因此，拟议的研究将促进鱼类生物学、免疫系统进化和DNA损伤/修复领域的知识。

The goal of this research program is to understand forms and functions of DNA damaging processes in fish. The human immune system uses DNA damage in the form of mutations as well as double strand DNA breaks (DSBs) to initiate the diversification of antigen receptor genes in lymphocytes. Our central hypothesis is that orthologous version of these DNA damaging enzymes in fish are involved in biological roles beyond the immune system, possibly in epigenetic genome reprogramming and development. All biological organisms have multiple DNA damage sensing, checkpoint and repair pathways. Consequently cells devote much energy to faithful maintenance and repair of their DNA sequence. In this light, the immune system of jawed vertebrates represents an exception. It is the sole example in all of biology where cells, in the course of their normal development and function, actively mutate and physically reorganize their own genomic DNA in a site-specific manner. The usage of DNA damage by immune systems is thought to have first appeared in cartilaginous fish. We have recently discovered that orthologous version of some DNA damaging enzyme involved in the human immune system have distinct activity profiles in specific fish species, and thereby mediate functions that are entirely outside the immune system. Based on this finding, we are proposing a new research program to broadly examine DNA damaging enzymes and their biological functions in the jawless vertebrate sea lamprey, sharks which are cartilaginous fish, and several species of bony fish including Atlantic cod, salmon and coelacanth. Our objectives are to 1) clone and characterize DNA damaging enzymes in these species, 2) examine tissue-specific expression patterns of these enzymes in response to immune stimuli and during development, 3) express, purify and examine the biochemical differences amongst fish DNA damaging enzymes, and 4) compare the biological functions of fish DNA damaging enzymes using genetically modified cells and transgenic animal models. If our hypothesis is correct, we expect to find that DNA damaging enzymes in some or all of these fish species have distinct tissue expression patterns and/or different biochemical properties as compared to their mammalian counterparts or each other. Our hypothesis also predicts that these enzymes can prompt different DNA damage responses and carry out divergent biological functions when expressed in cell lines or in transgenic animals.  This research program represents a new direction for my laboratory and we have initiated local, national and international collaborations with experts in fish biology and immunology. Regardless of whether results support our hypothesis, we will gain a comprehensive understanding of the forms and functions of fish DNA damaging enzymes. Thus, the proposed research will advance knowledge in the fields of fish biology, immune system evolution and DNA damage/repair.