de novo creation of base J in Leishmania

摘要 有20多种利什曼原虫属的病原是利什曼病的病原体。 利什曼病的症状包括自限性皮肤损害和内脏利什曼病，如果 不治身亡。在过去的5年里，全球有100多万例报告病例，其中20,000多例 每年报告的死亡人数。利什曼原虫生活在分支最深的真核生物谱系之一， 有许多独特和新颖的生物过程，其中两个对本提案很重要。这个 首先是蛋白质编码基因的组织，这些基因由RNA聚合酶II转录成长串 无关的基因。该基因簇从转录起始点转录为单一的初级RNA。 然后，转录通过多顺反子基因簇进行，单个mRNAs由 反式剪接，直到它到达转录终止位点。第二个过程是修改~1%的 胸苷(T)在脱氧核糖核酸中与葡萄糖形成β-D-葡萄糖-羟甲基尿嘧啶(J)。将T修改为J是a JBP1或JBP2对T进行羟基化的两步法。随后将葡萄糖添加到 通过一种特定的葡萄糖转移酶产生羟基。JBP1和JBP2在JJBP1的形成中起着不同的作用 负责在已经含有J的区域维持J，在新的DNA合成之后 形成的未经修饰的链。JBP2负责J的从头插入，主要是在JBP1错过的位点上，以及 最终决定哪些碱基被修饰为J。大多数J(~99%)定位于端粒，但J是 也在内部位点发现，最明显的是在除一个转录终止位点之外的所有位置。改进型的 从T到J在利什曼原虫中是必不可少的，因此对利什曼原虫如何决定哪些碱基有更深的理解 修饰将是开发潜在的靶向治疗的关键，该蛋白的产生 JBP2的序列不包含任何可以与DNA结合的基序，但它确实包含一种蛋白质 互动域。我们确定JBP2在一个复合体中与一种我们命名为J2TDP的蛋白质相互作用。这个 J2TDP的蛋白质序列包含一个Tudor结构域，这是一个已知与含有 二甲基赖氨酸或精氨酸，主要存在于组蛋白中。对J2TDP的识别使我们了解到 假设J2TDP结合了特定的组蛋白修饰，然后将JBP2招募到染色体上 修改相邻的TS以转换为J。在此建议中，我们将这一假设分解为以下三个部分 细分假说，然后测试假说的关键预测。1)J2TDP与组蛋白修饰结合。2) J2TDP将JBP2招募到染色体上。3)JBP2在染色体上的定位足以进行定位 对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.