de novo creation of base J in Leishmania

摘要 利什曼原虫属有超过20种致病性物种是利什曼病的病原体。 利什曼病的症状范围从自限性皮肤病变到内脏利什曼病，如果 不治疗。在过去5年中，全球报告的病例超过100万例， 每年报告的死亡人数。利什曼原虫存在于分支最深的真核细胞谱系之一， 拥有许多独特的和新颖的生物过程，其中两个是重要的这一建议。的 首先是RNA聚合酶II转录的蛋白质编码基因组织成长簇， 无关基因基因簇从转录起始位点转录为单个初级RNA。 然后，转录通过多顺反子基因簇进行，单个mRNA被 反式剪接，直到它到达转录终止位点。第二个过程是修改约1%的 DNA中的胸腺嘧啶（T）与葡萄糖形成β-D-葡糖基-羟甲基尿嘧啶（J）。T到J的修改是一个 通过JBP 1或JBP 2对T进行羟基化的两步法。随后将葡萄糖加入到 通过特异性葡糖基转移酶将羟基连接。JBP 1和JBP 2在J.JBP 1的形成中具有不同的作用。 负责在新的DNA合成后，在已经含有J的区域中维持J。 形成未修饰的链。JBP2负责J的从头插入，主要是在JBP1遗漏的位点， 最终决定哪些碱基被修饰为J。大多数J（~99%）位于端粒，但J 也发现在内部网站最显着的是在所有但一个转录终止位点。的修改 T到J在利什曼原虫中是必不可少的，因此更深入地了解利什曼原虫如何决定哪些碱基是 修饰将是至关重要的，在开发潜在的治疗靶向创建J。 JBP2的序列不包含任何允许它结合DNA的基序，但它确实包含一种蛋白质 交互作用域我们确定JBP2与我们命名为J2TDP的蛋白质在复合物中相互作用。的 J2TDP的蛋白质序列含有Tudor结构域，已知该结构域与含有 二甲基化赖氨酸或赖氨酸，其主要存在于组蛋白中。J2TDP的鉴定使我们 假设J2TDP结合特定的组蛋白修饰，然后将JBP2募集到染色体上， 修改相邻的Ts以转换为J。在本提案中，我们将此假设分解为以下三个 子假设，然后测试假设的关键预测。1)J2TDP结合组蛋白修饰。（二） J2TDP将JBP2募集到染色体上。3)将JBP 2定位于染色体上足以定位 修改J。

ABSTRACT There are over 20 pathogenic species of the genus Leishmania that are the causative agent for leishmaniasis. Symptoms for leishmaniasis range from self-limiting cutaneous lesion to visceral leishmaniasis that is fatal if left untreated. Globally there are more than 1 million reported cases in the last 5 years with over 20,000 fatalities reported annually. Leishmania resides in one of the most deeply branched eukaryotic lineages and harbors a number of distinct and novel biological processes, two of which are important for this proposal. The first is the organization of the protein coding genes transcribed by RNA polymerase II into long clusters of unrelated genes. The cluster of genes are transcribed as a single primary RNA from a transcription start site. Transcription then proceeds through the polycistronic cluster of genes with the individual mRNAs processed by trans-splicing until it reaches a transcription termination site. The second process is the modification of ~1% of thymidines (T) in DNA with glucose to form β-D-glucosyl-hydroxymethyluracil (J). The modification of T to J is a two-step process with the hydroxylation of T by either JBP1 or JBP2. Glucose is subsequently added to the hydroxyl group by a specific glucosyltransferase. JBP1 and JBP2 have distinct roles in the formation of J. JBP1 is responsible for maintenance of J, in regions already containing J, following DNA synthesis on the newly formed unmodified strand. JBP2 is responsible for de novo insertion of J, mostly at sites missed by JBP1, and ultimately determines which bases are modified to J. Most of J (~99%) is localized to the telomeres, but J is also found at internal sites most notably at all but one of the transcription termination sites. The modification of T to J is essential in Leishmania, so a deeper understanding of how Leishmania determines which bases are modified will be crucial in the development of potential therapeutic targeting the creation of J. The protein sequence of JBP2 does not contain any motif that would allow it to bind DNA, but it does contain a protein interaction domain. We determined that JBP2 interacts in a complex with a protein we named J2TDP. The protein sequence of J2TDP contains a Tudor domain, a motif known to interact with protein that contain dimethylated lysines or arginines, which are principally found in histones. The identification of J2TDP led us to the hypothesize that J2TDP binds a specific histone modification and then recruits JBP2 to the chromosome to modify adjacent Ts for conversion into J. In this proposal we break this hypothesis down in the follow three sub-hypotheses and then test key predictions of the hypotheses. 1) J2TDP binds a histone modification. 2) J2TDP recruits JBP2 to the chromosome. 3) Localization of JBP2 to the chromosome is sufficient for localized modification to J.